Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPI'ION
~, "METHOD FOR DETECTION AND TREATMENT OF BREAST CANCER"
TECHNICAL FIELD
.. 5 The present invention relates generally to methods of detection and diagnosis of
breast cancer and more particularly to a ~ gnc)stic method which relies on the
idPntifi~tion of marker genes ~ sed in pre-invasive cancers by microscopically-
directed cloning. Furthermore, this invention concerns the prevention, detection, and
diagnosis of breast cancer by addressing the molecular events which occur during the
earliest alterations in breast tissue.
The present invention also relates generally to methods of tre~tment of breast
cancer, and more particularly to gene therapy methods and methods for sclce,lillg
coln~ullds that induce ~A~s~ion of the BRCAl gene product.
~;
BACKGROUND ART
It will be appreciated by those sl~lled in the art that there exists a need for a
more sensitive and less invasive met=o of early detection and diagnosis of breast
cancer than those methods currently in use. Breast cancer plese.~Ls inherent difficulties
in regard to the ease with which it is llet~ctPd and diagnosed. This is in contrast to
detection of some other common cancers, inclllrling skin and cervical cancers, the latter
of which is based on cytomorphologic s~f~~ g techniques.
There have been several aLLen-~tS to develop improved mPtholls of breast cancer
detection and diagnosis. In the alLe---~ls to improve methods of detection and ~ no~i~
of breast cancer, numerous studies have searched for oncogene mutations, gene
amplification, and loss of heter~zygosiLy in invasive breast cancer (~ h~n, et al.,
1992; Cheickh, et al., 1992; Chen,et al, 1992; and, Lippman, et al, 1990). However,
few studies of breast cancer have analyzed gene mutations and/or altered gene
~ssion in ductal carcinoma in situ (DCIS). Investi~tors have demon~t~ted high
levels of pS3 protein in 13-40% of DCIS lesions employing a monoclonal antibody to
pS3, and subsequent seqU~ncin~ demonct~t~d mutations in several cases (Poller et al,
1992). The neu/erbB2 gene a~ to be amplified in a subset of DCIS lesions (Allredet al, 1992; Maguire et al, 1992). Histologic analysis of DCIS cases suggests that
mutations and altered gene expression events, as well as changes in chromatin and
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DNA content, occur predo.l.i~ ly in comedo DCIS (Bocker et al, 1992; Killeen et al,
1991; and, KO~ uw~ki et al, 1990), which has a rapid rate of local invasion and
progression to m~t~t~ Thus, there are p,csently no reliable marker genes for non-
comedo DCIS (NCDCIS, hereafter).
S Cancer in hllm~n~ appe~ to be a multi-step process which involves ~lug~cssion
from pre-m~lipn~nt to m~lign~nt to m~t~t~tic disease which ~lltim~ttoly kills the patient.
Epide.miologic studies in hllm~n~ have established that certain pathologic conditions are
"pre-m~lign~nt" because they are associated with increased risk of m~ n~ncy. There
is prece~ent for ~letecting and elimin~ting pre-invasive lesions as a cancer prevention
~Ll~y: dysplasia and carcinoma in-situ of the uterine cerviY are eY~mples of pre-
m~ n~ncies which have been succes~fully employed in the prevention of cervical
cancer by cytologic s.;,~enillg methods. Unfo,LunaLely, because the breast cannot be
sampled as readily as cervix, the development of scr~ning methods for breast pre-
m~lign~ncy involves more complex approaches than cytomorphologic screening now
~;ullcllLly employed to detect cervical cancer.
Pre-m~lign~nt breast disease is also characterized by an appalcnt morphological
~,og,cssion from atypical hyperplasias, to carcinoma in-situ (pre-invasive cancer) to
invasive cancer which ultim~tely spreads and met~t~i7~s resulting in the death of the
patient. Careful histologic t~ ";n~ion of breast biopsies has demon~tr~teA
intermPAi~te stages which have acquired some of these c~ cte~i~tics but not others.
Detailed epi-lemiological studies have established that dirrc,cllt morphologic lesions
progrcss at different rates, varying from atypical hyperplasia (with a low risk) to
comedo ductal carcinoma-in-situ which progresses to invasive cancer in a high
pcrcell~ge of p~ti~nt~ (London et al, 1991; Page et al, 1982; Page et al, 1985; Page
et al, 1991; and Page et al, 1978). Family history is also an il,l~,l~lt risk factor in
the development of breast cancer and increases the relative risk of these pre-m~lign~nt
lesions (Dupont et al, 1985; Dupont et al, 1993; and, London et al, 1991). Of
particular inlcresl is non-comedo carcinoma-in-situ which is ~soci~tPA with a greater
than ten-fold increased relative risk of breast cancer co~ cd to control groups
(Ottesen et al, 1992; Page et al, 1982). Two other reasons besides an increased relative
risk su~?poll the concept that DCIS is pre-m~lign~nt- 1) When breast cancer occurs in
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these patiPnt~ it regularly occurs in the same region of the same breast where the DCIS
was found; and 2) DCIS is frequently present in tissue a~jacent to invasive breast
cancer (Ottesen et al, 1992; Schw~ et al, 1992). For these reasons DCIS very likely
lcl~resellt~ a rate-limitin~ step in the development of invasive breast cancer in women.
DCIS (somPtimPs called intr~uct~l carcinoma) is a group of lesions in
which the cells have grown to completely fill the duct with pattern~ similar to invasive
cancer, but do not invade outside the duct or show met~t~es at presentation. DCIS
occurs in two forms: comedo DCIS and non-comedo DCIS. Comedo DCIS is often a
grossly palpable lesion which was probably considered "cancer" in the l9th and early
20th century and ~r~gr~sses to cancer (without definitive therapy) in at least 50% of
pati~nt~ within three years (Ottesen et al, 1992; Page et al, 1982). Most of themole~ul~r ~ltt~rati~ns which have been lc~ol~ed in pre-m~ n~nt breast disease have
been observed in cases of comedo DCIS (Poller et al, 1993; Radford et al, 1993; and,
Tsuda et al, 1993). Non-comedo DCIS is detected by microscopic analysis of breast
~spirates or biopsies and is a~oci~t~l with a 10 fold increased risk of breast cancer,
which collc~L~nds to a 25-30% absolute risk of breast cancer within 15 years (Ottesen
et al, 1992; Page et al, 1982; and, Ward et al, 1992).
Widespread application of mammography has changed the relative incidP.nce of
comedo and non-comedo DCIS such that NCDCIS now lep~csell~ the predoll~inan~
form of DCIS ~ gnose~l in the United States (Ottesen et al, 1992; Page et al, 1982; and
Pierce et al, 1992). Both forms of DCIS generally recur as invasive cancer at the same
site as the pre-m~ nant lesion (without der~-liv-e therapy). The l~lc;ul~or lesions to
DCIS are probably atypical ductal hy~ lasia and prolir~.~live disease without atypia
which are associated with lower rates of breast cancer development, but show further
increased risk when ~soci~t~d with a family history of breast cancer (Dupont et al,
1985; Dupont et al, 1989; Dupont et al, 1993; Lawrence, 1990; London et al, 1991;
Page et al, 1982; Page et al, 1985; Page et al, 1991; Page et al, 1978; Simpson et al,
1992; Solin et al, 1991; Swain, 1992; Weed et al, 1990).
What is nffllecl, then, is a sensitive method of dete~tion and diagnosis of breast
cancer when the cancerous cells are still in the pre-invasive stage. To ill~ t~ the
usefulness in early breast cancer detection of a marker gene and its encoded protein,
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con~ider the dramatic impact that p-osl~te specific antigen has had on early stage
prostate cancer. This method of early dete~tion and diagnosis of breast cancer is
presenlly lacking in the prior art.
Breast cancer occurs in hereditary and sporadic forms. Recently the BRCA 1
gene has been cloned and shown to be m~lt~ted in kindreds with hereditary breast and
ovarian cancer (Hall et al. 1990, Miki, Y. et al. 1994, Friedman et al. 1994, Castilla
et al. 1994, Simard et al. 1994). Although 92% of f~milies with two or more cases of
early-onset breast cancer and two cases of ovarian cancer have germ-line mutations in
BRCA 1 (Narod et al. in press), the gene has not been shown to be mutated in anytruly sporadic case to date (Futreal et al. 1994). Despite the surprising paucity of
som~tic~lly acquired mutations in sporadic breast cancer, it is still a likely tumor
su~lessor gene with a key role in breast epithelial cell biology. The BRCA 1 gene
encodes a protein of 1863 amino acids with a predicted zinc finger domain observed
in proteins which regulate gene transcription. Until the discovery of the function of the
BRCAl gene in conjucntion with the delopment of the present invention, the function
was unknown.
DISCLOSIJRE OF l~ INVENTION
FridPminlc)gic studies have established that NCDCIS of the breast is associated
with a ten-fold increased risk of breast cancer (absolute risk of 25-30%). It seems
likely that this pre-invasive lesion is a dele~ te pL~iulsor of breast cancer because
the subsequent development of breast cancer is regularly in the same region of the same
breast in which the NCDCIS lesion was found. Illl~.L~t aspects of the present
invention concern i.~ol~tYl DNA segmpnt~ and those isolated DNA se~mPnt.~ inserted
into recombinant vectors encoding dirr~Le"tially ~;A~.essed marker genes in abnormal
tissue, specifically in NCDCIS, as colll~ar~d with those expressed in normal tissue, and
the creation and use of recombinant host cells through the application of DNA
technology, which express these dirre elltially eAp.essed marker genes (Sambrook et al,
1989).
RP~ P~ there are no cell lines or animal models which clearly display known
çh~r~ctPri~tics of pre-invasive breast disease, human breast tissue s~mrles are e~enti~l
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for studying pre-invasive breast di~e. Using human tissue ~mpltqs, we subsequently
have developed a method for cDNA cloning from histologically i~lentified lesions in
human breast biopsies. We have used this method to clone genes which are
~ dirr~enLially ~A~essed in pre-invasive breast lesions such as NCDCIS lesions as
S colllp~c;d to genes ~ .essed in normal tissue. The dirr~t;lltially ~Apr~ssed genes
det~ted in pre-invasive breast cancer are called marker genes. T~entific~tic)n of marker
genes for pre-invasive breast disease provides i~ uved methods for detection andnosi~ of pre-invasive breast cancer tissue, and further provides marker genes for
studies of the molecular events involved in proglt;ssion from pre-invasive to m~ n~nt
breast (~ e
Analysis of marker gene ~Al,le~sion in NCDCIS p-esel.ts the advantage that
cancerous breast tissue at that stage is non-invasive. Detection and diagnosis of
NCDCIS by means of dirr~ ~ntially ~A~ressed marker genes col--p~d to the same
marker genes in normal breast tissue, would allow a greater ability to detect, prevent
and treat the disease before it becomes invasive and met~t~i7~s. The stage or
intermeAi~t~- condition of NCDCIS is a particularly good c~n~ te for early
intervention because it is 1) prior to any invasion and thus prior to any threat to life;
2) it is followed by invasive carcinoma in over 30% of cases if only treated by biopsy;
and, 3) there is a long "window" of ol)~ lu--ily (4-8 years) a~-u~ .oly before
invasive neoplasia occurs. Thus, NCDCIS is an ideal target for early diagnosis. While
these morph-llogically ~ fin~d int~rm~i~te endpoints have been widely accepted,
plOgl~SS in ~lefining the rnolec~ r coll~lal~s of these lesions has been halll~Led by an
inability to identify and sample them in a manner which would allow the appli~tion of
molecular techniques.
Frozen tissue blocks from breast biopsies were used to construct and screen
cDNA libraries pl~p~cd from NCDCIS tissue, normal breast tissue, breast cancer
tissue, and normal human breast epitheli~l cells. Several cDNAs which were
differentially eA~lessed in human DCIS epith~ l cells co~ ared to normal breast
epithelial cells were cloned and sequenced. One gene which is diLrerentially expressed
is the M2 subunit of RibRed which is e~ ssed at low levels in human breast epithPli~l
cells but at higher levels in 4 out of 5 DCIS tissue samples. It is prçs~-m~d that the
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altered morphologic appe~d,~ce and dele~ nt biologic behavior of DCIS results from
altered expression of genes (such as RibRed) which is important in the inlluction of
breast cancer in h~lm~n~
This invention, therefore, provides a method of detecting and ~ gnosing pre-
S invasive breast cancer by analyzing marker genes which are dirrelentially GA~ressed in
non-comedo DCIS cells. Histopathologic studies have demon~t~ted that these
morphologic p~ttern~ in breast tissue lead to invasive breast cancer in at least 20-30%
of p~ti~nt~. The present method analyzes gene GAyrGssion in normal, pre-m~lign~nt and
m~lign~nt breast biopsies; and, it allows ~imlllt~neous co~p~ on and cloning of
marker genes which are dirrelentially GAyressed in pre-invasive breast cancer. These
marker genes can then be used as probes to develop other diagnostic tests for the early
cletection of pre-invasive breast cancer.
The present invention concerns DNA segm~nt~ ol~t~kle from both normal and
abnormal human breast tissue, which are free from total genomic DNA. The isolated
DCIS-l protein product is the regulatory e1pm~nt of the RibRed enzyme. This and all
other isolatable DNA segm~nt~ which are dirrerGnlially GAy~ssed in preinvasive breast
cancer can be used in the detection, diagnosis and tre~tnlpnt of breast cancer in its
earliest and most easily treatable stages. As used herein, the term "abnormal tissue"
refers to pre-invasive and invasive breast cancer tissue, as eYPmrlifiP~ by collected
samples of non-comedo or comedo DCIS tissues.
As used herein, the term "DNA segmPnt" refers to a DNA molecule which has
been isolated free of total genomic DNA of a particular speciPs Therefore, a DNAsegment encoding a dirre~e~llially G~ GssGd protein (as measured by the eA~l~;ssion of
mRNA) in abnormal tissue refers to a DNA segm~-nt which contains dirrG t;,llially
G~rGssed-coding sequences in abnormal tissue as colllpaLGd to those e~essed in
normal tissue, yet is isolated away from, or purified free from, total genomic DNA of
Homo sapiens sapiens. Furthermore, a DNA se.gmPnt encoding a BRCAl protein
refers to a DNA .segmPnt which contains BRCAl coding sequences, yet is i~ ted away
from, or purified free from, total genomic DNA of Homo sapiens sapiens. Tncl~l~le~
within the term "DNA segmPnt", are DNA segmPnt~ and smaller fr~gm~nt~ of such
se.gmPnt~, and also recombinant vectors, inclll-ling, for PY~mple, pl~mi~ls, cosmids,
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phage, viruses, and the like.
Similarly, a DNA segmPnt compri~in~ an i~ol~t~l or purified diLreLe,ltially
~A~lcssed gene or compri~ing an icol~tP~ or purified BRCA1 gene refers to a DNA
r se~mPnt incl~lrling dirrele ~lially ~A~l~;ssed coding sequences or BRCA1 coding
sequences isolated subst~nti~lly away from other n~tllr~lly occurring genes or protein
encoding sequences. In this respect, the term "gene" is used for .cimplicity to refer to
a functional protein, polypeptide or peptide ent~in~ unit. As will be understood by
those in the art, this functi()n~l term inclll~es both genomic sequences and cDNA
sequences. "T~ol~tPA subst~nti~lly away from other coding sequences" means that the
gene of interest, in this case, any dirrerenlially ~Lplessed marker gene or the BRCA1
gene, forms the ~ignifi~nt part of the coding region of the DNA segm~nt and that the
DNA se.~ment does not contain large portions of naturally-occu~ g coding DNA, such
as large chromosomal fr~gm~nt~ or other functi~-n~l genes or cDNA coding regions.
Of course, this refers to the DNA sepm~nt as originally i~ol~t~, and does not eYcll~de
genes or coding regions later added to the segment by the hand of man.
In particular emb~lim~nt~, the invention concerns i~ol~ted DNA segm~ont~ and
recombinant vectors inccl~ldli~lg DNA sequences which encode dirrelenlially
t;~ressed genes in pre-invasive breast cancer, each which inçllldes within its amino
acid sequence an amino acid sequence in accor~ance with SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID
NO:7, all seq id no:s 1-7 are derived from non-comedo DCIS samples from Homo
sapiens sapiens. In other particular embo lim~-nt~, the invention conr~rn~ isolated DNA
se.gm~nt~ and recombinant vectors inccl~ldting DNA sequences which encode the M2subunit of human RibRed that incl~ldes within its amino acid sequence the similar amino
acid sequence of h~m~hr RibRed coll~s~nding to the M2 subunit of h~m~tor RibRed.In certain embo~ , the invention concerns i~ol~ted DNA segments and
recombinant vectors which partially or wholly encode a protein or peptide that inrlndes
within its amino acid sequence an amino acid sequence e~sPnti~lly as partially or wholly
encoded, res~ ely, by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQID NO:5, SEQ ID NO:6, or SEQ ID NO:7. Naturally, where the DNA
segm~nt or vector encodes a full length dirrt;lel-lially ~ressed protein, or is int~n~ed
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for use in ~ essillg the dirr~lel~tially expressed protein, the most ~rerellc;d sequences
are those which are eccPnti~lly as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 and which
encode a protein that exhibits dirrer~lllial expression, e.g., as may be dele~ in~A by the
S dirrelel~lial display or dirrelu~tial sequencing assay, as disclosed herein.
The term "a sequence escenti~lly as set forth in SEQ ID NO:1, SEQ ID NO:2,
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7"
means that the sequence subst~nti~lly corresponds to a portion of SEQ ID NO: 1, SEQ
ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID
NO:7, respectively, and has relatively few nucleotides which are not idPntic~l to, or a
biologically functional equivalent of, the nucleotides of the respective SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ
ID NO:7. The term "biologically functional equivalent" is well understood in the art
and is further defined in detail herein, for example see pages 24 through 25.
Accordingly, sequences which have between about 70% and about 80%; or more
preferably, between about 81% and about 90%; or even more preferably, between
about 91% and about 99%; of amino acids which are identir~l or functi~n~lly
equivalent to the amino acids of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ
ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 will be sequences which
are "e~-nti~lly as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7", respectively.
In particular embolim~nt~, the invention concerns a drug screening mPthod and
a gene therapy method that use isolated DNA segments and recombinant vectors
incorporating DNA sequences which encode a protein that includes within its amino
acid sequence an amino acid sequence in accordallce with SEQ ID NO:49, SEQ ID
NO:49 derived from breast tissue from Homo sdpiens. In other particular
emb~liment~, the invention concerns i~ol~t~d DNA sequences and recombinant DNA
vectors incc~ dLing DNA sequences wich encode a protein taht includes with its
amino acid sequence the amino acid sequence of the BRCA1 gene product from humanbreast tissue.
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In certain emb~limtont~ the invention concerns methods using i~ol~ted DNA
segments and recombinant vectors which partially or wholly encode a protein or peptide
that inrll~des within its amino acid sequence an amino acid sequence es~-nl;~lly as set
forth in SEQ ID NO:49. Naturally, where the DNA segment or vector encodes a fulllength BRCAl protein, or is intended for use in ~A~les~ g the BRCA1 protein, themost plcr~llcd sequences are those which are çssenti~lly as set forth in SEQ ID NO:47
and which encode a protein that retains activity as a negative growth regulator in human
breast cells, as may be detPrmin~d by ~nti~n~e assay, as r1i~rloserl herein.
The term "a sequence es~.ti~lly as set forth in SEQ ID NO:1, SEQ ID NO:2,
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7"
means that the sequence ~ubs~ 11y collcspollds to a portion of SEQ ID NO: l, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:S, SEQ ID NO:6, or SEQ ID
NO:7, respectively, and has relatively few nucleotides which are not identi~l to, or a
biologir~lly functional equivalent of, the nucleotides of the respective SEQ ID NO:l,
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:S, SEQ ID NO:6, or SEQ
ID NO:7. The term "biologically functional equivalent" is well understood in the art
and is further ~lefinrd in detail herein, for ~qY~mrle see pages 24 through 25.
Accordingly, sequences which have betwecll about 70% and about 80%; or more
preferably, belw~n about 81% and about 90%; or even more preferably, bclw~n
about 91% and about 99%; of amino acids which are identir~l or f~mrti-)n~lly
equivalent to the amino acids of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ
ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 will be sequences which
are "e~se~ lly as set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7", lc~ ely.
The term "a sequence esse~ lly as set forth in SEQ ID NO:49" means that the
sequence subst~nti~lly corr~-~ron-l~ to a portion of SEQ ID NO:49 and has relatively
few amino acids which are not idenhr~l to, or a biologically functional equivalent of,
the nucleotides of SEQ ID NO:49. The term "biologically functional equivalent" is
well understood in the art and is further defined in detail herein, for ~Y~mple see pages
24 through 25. Accordingly, sequences which have between about 70% and about
80%; or more preferably, between about 81% and about 90%; or even more
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preferably, belweell about 91% and about 99%; of amino acids which are i~lentir~l or
functionally equivalent to the amino acids of SEQ ID NO:49 will be sequences which
are "es~enti~lly as set forth in SEQ ID NO:49".
In certain other embo limPnt~, the invention concerns isolated DNA segments
and recombinant vectors that include within their sequence a nucleic acid sequence
essP-nti~lly as set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7. The term "es~Pnti~lly as set forth
in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ
ID NO:6, and SEQ ID NO:7" is used in the same sense as described above and meansthat the nucleic acid sequence subst~nti~lly corresponds to a portion of SEQ ID NO: 1,
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ
ID NO:7, respectively, and has relatively few codons which are not i(lPnti~l, orfunctionally equivalent, to the codons of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3,
SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively.
Again, DNA se.gmPnt~ which encode proteins exhibiting dirr~rential expression will be
most ~lc;relled. The term "functionally equivalent codon" is used herein to refer to
codons that encode the same amino acid, such as the six codons for arginine or serine,
and also refers to codons that encode biologically equivalent amino acids (see Figure
8).
In certain other embodimPnts, the invention concerns a method for scr~el~illg
drugs and a gene therapy method which involve the use of i~ol~tP~ DNA segmPnt~ and
recombinant vectors that include within their sequence a nucleic acid sequence
essPnti~lly as set forth in SEQ ID NO:47 and SEQ ID NO:48. The term ''e~sPnti~lly
as set forth in SEQ ID NO:47 and SEQ ID NO:48" is used in the same sense as
described above and means that the nucleic acid sequence subst~nti~lly corresponds to
a portion of SEQ ID NO:47 and SEQ ID NO:48 respectively, and has relatively few
codons which are not identical, or function~lly equivalent, to the codons of SEQ ID
NO:47 and SEQ ID NO:48, respectively. Again, DNA se~mPnt~ which encode
proteins exhibiting the negative regulatory activity of the BRCAl will be most
~r~f~lled. The term "functionally equivalent codon" is used herein to refer to codons
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that encode the same amino acid, such as the six codons for arginine or serine, and also
refers to codons that encode biologically equivalent amino acids (see Figure 8).It will also be understood that amino acid and nucleic acid sequences may
include additional re~i~ues, such as additional N- or C-telnlillal amino acids or 5' or
3' sequences, and yet still be essçnti~lly as set forth in one of the sequences disclosed
herein, so long as the sequence meets the criteria set forth above, incl~lrling the
intton~llce of biological protein activity where protein ~;~ression is concerned. The
addition of le~ l sequences particularly applies to nucleic acid sequences which may,
for example, include various non-coding se~uences fl~nking either of the 5' or 3'
portions of the coding region or may include various intPrn~l sequences, i.e., introns,
which are known to occur within genes.
Excepting intronic or fl~nking regions, and allowing for the degeneracy of the
genetic code, sequences which have between about 20% and about 50%; or more
preferably, between about 50% and about 70%; or even more preferably, between
about 70% and about 99%; of nucleotides which are itle-nti~l to the nucleotides of SEQ
ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, and SEQ ID NO:7will be sequences which are "çssenti~lly as set forth in SEQ
ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, and SEQ ID NO:7", respectively. Sequences which are es~Pnh~lly the same asthose set forth in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ
ID NO:5, SEQ ID NO:6, and SEQ ID NO:7 may also be functionally ~fin~ as
sequences which are capable of hybri~li7.ing to a nucleic acid segment col,t;.;ni~-g the
comr)lemPnt of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, under relatively stringent
conditions. Suitable relatively stringent hybri~i7~tion conditions will be well known to
those of skill in the art (Sambrook et al, 1989).
Excepting intronic or fl~nking regions, and allowing for the degeneracy of the
genetic code, sequences which have between about 20% and about 50%; or more
preferably, between about 50% and about 70%; or even more preferably, between
about 70% and about 99%; of nucleotides which are identi~l to the nucleotides of SEQ
ID NO:47 and SEQ ID NO:48will be sequences which are "es~Pnti~lly as set forth in
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SEQ ID NO:47 and SEQ ID NO:48", respectively. Sequences which are e~çnti~lly
the same as those set forth in SEQ ID NO:47 and SEQ ID NO:48 may also be
function~lly defined as sequences which are capable of hybridi7ing to a nucleic acid
se.~mPnt con~ ing the complement of SEQ ID NO:47 and SEQ ID NO:48,
respectively, under relatively stringent conrlition~. Suitable relatively stringent
hybri~li7~tion conditions will be well known to those of skill in the art (Sambrook et al,
1989).
It is also i~ t to understand the molecular events which lead to progression
from pre-invasive to invasive breast cancer. Breast cancer is a disease that is presllm~d
to involve a series of genetic alterations that confer increasing growth indepPndence and
met~.~t~tic capability on somatic cells. Identifying the molecular events that lead to the
initial development of a neoplasm is th~ero~ critical to underst~nding the filntl~ment~l
merl~ni~m~ by which tumors arise and to the selection of optimal targets for gene
therapy and chemopreventive agents. As intermPAi~te endpoints in neoplastic
development, some pre-m~lign~nt breast lesions represent in.~,~t, and possibly
rate-limiting steps in the progression of human breast cancer, and careful
epidPmiolngical studies have established the relative risk for breast cancer development
for specific histologic lesions. In particular, invasive breast cancer develops in the
region of the previous biopsy site in at least 25-30% of p~tient.~ following diagnosis of
non-comedo DCIS providing strong evidence that this pre-m~ n~nt lesion is a
dele""in~llt event in breast cancer progression. While these morphologically defined
intern~ te endpoints have been widely accepted, progress in defining the molecular
correlates of these lesions has been hampered by an inability to identify and sample
them in a manner which would allow the application of mohP~ r techniques.
The present invention incllldes a co."~ on of gene t;~les~ion belv~~n
multiple breast tissue biopsy ~mples as a means to identify dirr~lcl~lially t;~lt;ssed
genes in pre-m~lign~nt breast disease colllp~d with normal breast tissue. These
genetic m~ should be extremely useful reagents for early diagnosis of breast
cancer, and for the dPlinP~tion of molecular events in progression of breast cancer.
TdPnti~ tion of gene l.l~h~l~ which are expressed in the majority of pre-
invasive breast cancer tissue ~mples involves cDNA library plepa dlion from both
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normal and abnormal tissue. This is followed by either a modified dirr~cl~ial display
method or a differential screening method to identify differential expression of genes
which is subsequently confirm~d by RT-PCR, nllcl~ ion assays and in situ
hybridi7~tion of DCIS tissue RNA and control tissue RNAs (Sambrook et al, 1989).Use of genetic PnginPPring metho l~ can bias the sc ~ning to specifi~ y identify genes
whose encoded proteins are secreted or are present at the cell surface, in order to find
proteins which will be useful ~ krl.~i for diagnostic blood tests (secreted proteins) or
for diagnostic im~ging studies (cell surface proteins).
Thus, the method of the present invention begins with the collection of at leastone tissue sample by a microscopically-directed collection step in which a punch biopsy
is obtained exclusively from abnormal tissue which eYhibit~ histological or cytological
~h~r~ctPri~tics of pre-invasive breast cancer. Preferably, the sample site will be an
i~ol~t~hle tissue structure, such as ductal epithelial cells from pre-invasive breast
cancer tissue. The mRNA is purified from the sample. Then, a cDNA library is
~ ed from the mRNA purified from the abnormal tissue sample (Sambrook et al,
1989).
A normal tissue sample is then obtained from the patient, using a sample site
from an area of tissue which does not exhibit histological or cytological char~ctPri~ti~s
of pre-invasive cancer. A cDNA library is also p~ep~ed from this normal tissue
~0 sample.
The abnormal tissue cDNA library can then be col,lpa~ed with the normal tissue
cDNA library by dirr~; t;lltial display or dirrerenlial screening to delç~ ",ine whether the
e;Al~le~sion of at least one marker gene in the abnormal tissue sample is dirr~;~ei~l from
the ~;Apression of the same marker gene in the normal tissue sample.
~5 Further di~gnostic steps can be added to the mPthod by cloning the marker gene
using sequence-based ~mplific~tiQn to create a cloned marker gene which can then be
DNA-sequenced in order to derive the protein sequence. The protein sequence is then
used to generate antibodies which will recognize these proteins by antibody lecognilion
of the antigen. The presence of the antibody-recognized antigen can then be dete~tPd
~0 by means of conventional medical diagnostic tests.
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14
This invention also includes methods of scr~lung for compounds and gene
therapy methods using t,he BRCAl gene. BRCAl mRNA is expressed at 5-10 fold
higher levels in normal ~ tissue than in invasive breast cancer samples.
Having de,mon~tr~t~ that mRNA ~ ession levels of BRCAl are higher in normal
S Ill~."",~,.y cells than in cancer cells, ~nti~Pn~e methods were used to test the hypothesis
that BRCAl ~ression inhibits cell growth. These tests showed that ~iimini.~h~d
~ession of BRCAl increased the proliferative rate of breast cells.
An object of the present invention, then, is to provide a method of early
de,tection of pre-invasive breast cancer in human tissue.
It is a further object of this invention to identify early marker genes for pre-invasive breast disease which can be used in screening methods for early pre-invasive
breast cancer.
It is also an object of this invention to produce a cDNA library from pre-
invasive breast cancer tissue r~slllting in a perm~nPnt genetic sample of that pre-
invasive breast cancer tissue.
It is also an object of this invention to provide a drug or biological screeningmethod using the BRCA 1 prollloler region and gene therapy method using the BRCA 1 gene.
List of Abbreviations
TPA Phorbol 12-myristate 13-acetate
MCF-7 An immortalized cell line derived from a mPt~ct~ of
human breast cancer
HMEC A primary (non-immortalized) cell line derived from
breast epithelial cells obtained during re~luc.tion
mammoplasty
DCIS Ductal Carcinoma-in-situ
NCDC Non-Comedo Ductal Carcinoma in situ
cDNA Compl~,m~nt~ry DNA obtained from an RNA template
DNA Deoxyribonucleic Acid
RT-PCR Reverse Transcriptase-Polymerase Chain Reaction
RibRed Ribonucleotide I2educt~e
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Fig. 1 shows Table I which describes ana~ ic lesion types in the human breast
with pre-m~lign~nt implir~ticm
Fig. 2 shows a model for pre-m~lign~nt conditions, hi~hlighting m~gnitu-le of
risk for progression to clinical m~lign~ncy.
Fig. 3 CCll~ S color photos of DCIS tissue, before (upper left panel) and after
m-i~;roscopically-directed excisional punch biopsy (upper right panel). The lower panels
show tissue samples of normal breast tissue (lower left panel), and invasive breast
cancer (lower right panel).
Fig. 4 shows expression of collagen III mRNA in tissue mRNA ~mI)les,
analyzed by RNase protection assay methods.
Fig. 5 shows dirr~len~ial display of cDNAs obtained from patient tissue ~mple~
and controls.
Fig. 6 shows a co~ on of the se~quence between DCIS-l and the human and
h~m~tPr genes.
Fig. 7 shows ~;A~lession of DCIS-l m--RNA in tissue mRNA samples analyzed
by RNase protection assay as described in the legend to Figure 4.
Fig. 8 is Table II which displays the genetic code.
Fig. 9 is a Table which lists differentially t;~lcssed marker genes.
Figs. lOA and lOB shows ~;;A~lcssion of BRCAl mRNA during breast cancer
~lu~lcs~ion by PCR det~Pction and nuc~ r~ ion assay, lcs~ec~ ely.
Figs. llAand llBisacc.np~ onofBRCAl ~;A~lessioninnormalbreastand
invasive breast cancer using nuclease protection assay of RNA, respectively.
Figs. 12A, 12B, and 12C show that ~nti~Pn~P inhibition of BRCAl ~ccP-ler~tPs
Ill~lnllli1ly cell prolife~tion.
Figs. 13A and 13B includes a Northern blot of mRNA and nuclear runon studies
that show that ribonucleotide reduct~e M2 mRNA is cell cycle regulated in MCF-7
cells.
Fig. 14 includes a nll( lP~e pl~leclion assay that shows that ~nti~nse inhihition
of BRCAl in human ~ r cells decreases BRCAl mRNA and increases
ribonucleotide recluct~e mRNA.
.
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16
UTILlIY STATEMENT
The detection of dirr~le~ lially expressed genes in pre-invasive breast tissue,
specifically in non-comedo ductal carcinoma in situ as co~ ared to genes expressed in
normal tissue, is useful in the diagnosis, prognosis and tre~tm~-nt of human breast
cancer. Such di~r~,elllially expressed genes are effective marker genes in 1i~ting the
~ignific~ntly increased risk of breast cancer in a patient expressing these differentially
~,essed marker genes. These marker genes are useful in the detection, early
diagnosis, and tre~tm~nt of breast cancer in humans.
The discovery of the function of the BRCA 1 gene has broad utility inclll-iing,
in the present invention, development of methods to treat f~mili~l and sporadic breast
cancers as well as screen for thel~peulic drugs through production of illlpo
indic~tor compounds.
A~ llVll ~ STAI~MENT
Of the ~lirrerc~ntially eA~l~ssed genes described in this invention, DCIS-l
encodes a gene similar to the M2 subunit of h~m~t~r ribonucleotide redl~cP~e. The
M2 subunit of ribonucleotide reduct~e (RibRed, hereafter) is responsible for regulation
of RibRed. The differential levels of ~A~ltssion of the marker genes described in this
invention (Seq ID No.s 1-7), intlic~tP genetic changes which have been linked to the
presence of pre-invasive breast cancer.
The BRCAl gene (Seq. ID No. 47) is dirrel~l~lially ~Apressed in invasive breast
cancer cells. The BRCAl gene product is a negative regulator of m~mm~ry cell
proliferation which is ~A~l~ssed at dimini~hed levels in sporadic breast cancer.
BEST MODE FOR CARRYING OUT THE INVENTION
For the purposes of the subsequent description, the following definitions will be
used:
Nucleic acid sequences which are "complementary" are those which are capable
of base-pairing according to the standard Watson-Crick complemPI~t~ y rules. That
is, that the larger purines will always base pair with the smaller pyrimi~lin~ to form
only combinations of Guanine paired with Cytosine (G:C) and Adenine paired with
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17
either Thymine (A:T) in the case of DNA or .AdeninP paired with Uracil (A:U) in the
case of RNA.
"Hybri-1i7~tion techniques" refer to molecular biological techniques which
involve the binding or hybritli7~ticln of a probe to complemPnt~ry sequences in a
polynucleotide. Tncluded among these techniques are northern blot analysis, southern
blot analysis, nuclease p oleclion assay, etc.
"Hybrilli7~ti--n" and "binding" in the context of probes and denatured DNA are
used intelchal~geably. Probes which are hybridi_ed or bound to denatured DNA areag~.~ga~ed to complemPnt~ry sequences in the polynucleotide. Whether or not a
particular probe remains aggregated with the polynucleotide depends on the degree of
complçmPnt~rity, the length of the probe, and the stringency of the binding conditions.
The higher the stringçnsy~ the higher must be the degree of complem~ y and/or the
longer the probe.
"Probe" refers to an oligonucleotide or short fragment of DNA dP~ignPd to be
snffici~,ntly complem~nt~ry to a sequence in a denatured nucleic acid to be probed and
to be bound under selected stringency c~ n~itiQnS.
NLabel" refers to a m~lifi~tion to t,he probe nucleic acid t,hat enables t,he
eXperimpntpr to identify the labeled nucleic acid in the presence of unlabeled nucleic
acid. Most commonly, this is the rep1~~~,mP,nt of one or more atoms with r~flio~,tive
isotopes. However, other labels include covalently ~tt~'hP,d cl~ ~ upllores, fluorescent
moeities, enzymes, ant,igens, groups with sperific react,ivity, chPmilllminPscent
moeities, and electrochpmi~lly detect~hle moeities, etc.
"Marker gene" refers to any gene selPr~ted for detection which displays
dirre~ tial eA~lession in abnorrnal tissue as opposed to normal t,issue. It is also
referred to as a dirr~ ially eA~lessed gene.
HMarker prot,ein" refers to any protein encoded by a "marker gene" which
protein displays dirrelen~ial ~Aples~ion in abnormal tissue as opposed to normal t,issue.
''Ti~uemi7Pr~ describes a tissue homogçni7~tion probe.
"Abnormal tissue" refers to pathologic tissue which displays cytologic,
histologic and other de-fining and derivative features which differ from that of normal
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18
tissue. This inclll(les in the case of abnormal breast tissue, among others, pre-invasive
and invasive neoplasms.
"Normal tissue" refers to tissue which does not display any pathologic traits.
"PCR technique" describes a method of gene amplific~tion which involves
S sequenced-based hybritli7~tinn of primers to spe~ific genes within a DNA sample (or
library) and subsequent ~mplifi~tiQn involving multiple rounds of ~nnP~ling, elong~tiQn
and dP-n~tur~tiQn using a heat-stable DNA polymPr~e.
"RT-PCR" is an abbreviation for reverse transcriptase-polymerase chain
reaction. Subjecting mRNA to the reverse tr~n~criI)tase enzyme results in the
production of cDNA which is compl~-mPnt~ry to the base sequences of the mRNA.
Large amounts of selected cDNA can then be produced by means of the polymerase
chain reaction which relies on the action of heat-stable DNA polymerase produced by
Thermus aquaticus for its amplific~ticm action.
"Microscopically-directed" refers to the method of tissue s~mpling by which the
tissue .~mpled is viewed under a microscope during the sampling of that tissue such
that the sampling is precisely limited to a given tissue type, as the investig~tQr requires.
Specifically, it is a collection step which involves the use of a punch biopsy inst~ument.
This surgical instrument is stereot~tic~lly ma=nually-directed to harvest exclusively from
abnormal tissue which exhibits histologic or cytologic char~ct~ ti~s of pre-invasive
cancer. The harvest is correlated with a col"~al~ion slide, stained to recognize the
target tissue.
"Differential display" describes a method in which ~ressed genes are
co~ ~ed between ~mples using low stringency PCR with random oligonucleotide
primers.
"Differential screening" describes a m~tho~ in which genes within cDNA
libraries are co~ ~ed between two samples by dirr~elltial hybridization of cDNAs to
probes pl~ d from each library.
"Nuclease protection assay" refers to a method of RNA (lu~ ;nn which
employs strand specific nuc~ es to identify spe~ific RNAs by detection of duplexes.
"Dirre~lllial e~lc;ssion" describes the phenomenon of dirrereiltial genetic
e~ ssion seen in abnormal tissue in co",p~ on to that seen in normal tissue.
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19
~T.~ol~t~ble tissue structure" refers to a tissue structure which when vi~u~li7Pli
microscopically or otherwise is able to be isolated from other dirrelei~t ~ullounding
tissue types.
"In situ hybri~i7~tion of RNA" refers to the use of labeled DNA probes
employed in conjunction with histological sections on which RNA is present and with
which the labeled probe can hybridize allowing an investig~tor to vim~li7e the location
of the specific RNA within the cell.
"Comedo DCIS cells" refers to cells compri~ing an in situ lesion with the
combined fealules of highest grade DCIS.
"Non-comedo DCIS cells" refers to cells of DCIS lesions without comedo
realules.
"Cloning" describes sep~.~t;on and i~ol~tion of single genes.
"Seqllencing" describes the de~....in~l;on of the sper.ific order of nucleic acids
in a gene or polynucleotide.
The present invention provides a method for detecting and diagnosing cancer by
analyzing marker genes which are differentially t;~cssed in early, pre-invasive breast
cancer, sperifir~lly in non-comedo DCIS cells. Our histopathologic studies have
demon.~tr~ted that certain morphologic p~ttern.~ in breast tissue are pre-m~lign~nt,
leading to invasive breast cancer in at least 20-30% of p~ti~nt~. We have developed
a new method for analyzing gene ~A~resi,ion in normal, pre-m~lign~nt and m~lign~nt
breast biopsies which allows ~imlllt~neous co---~ on and cloning of marker geneswhich are dirr~ell ially e~lcssed in pre-invasive breast cancer. These marker genes
(which appear as dirr~lelllially ~A~ressed genes in pre-invasive breast cancer) can be
used as probes to develop diagnostic tests for the early detection of pre-invasive breast
cancer (Sambrook, 1989).
The present invention thus compri.~C, a method of identifir~tion of marker geneswhich are e~lessed in the majority of pre-invasive breast cancer tissue samples. It
involves cDNA library pl~ on followed by a modified dirr~relltial display method.
Use of genetic rngin~ring methods (Sambrook, 1989) can bias the screening to
sper-ific~lly identify genes whose encoded proteins are secreted or are present at the cell
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surface, in order to find proteins which will be useful markers for diagnostic blood tests
(secreted proteins) or for diagnostic im~ging studies (cell surface proteins).
Naturally, the present invention also encomp~ses DNA segm~nt.~ which are
comple",~ , or es~-nti~lly complP-mPnt~ry, to the sequence set forth in SEQ ID
NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:47 and SEQ ID NO:48. Nucleic acid sequences which are
"complem~nt~ry" are those which are capable of base-pairing according to the standard
Watson-Crick complem~ntarity rules. As used herein, the term "complem~nt~ry
sequences" means nucleic acid sequences which are subst~nti~lly complementary, as
may be ~s~ed by the same nucleotide col"~ on set forth above, or as defined as
being capable of hybridizing to the nucleic acid segmt~-nt of SEQ ID NO:l, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:47 and SEQ ID NO:48 under relatively stringent col flitionc such as those
described herein.
The nucleic acid segment~ of the present invention, regardless of the length of
the coding sequence itself, may be combined with other DNA sequences, such as
promoters, polyadenylation ~ign~ lrlition~l restriction enzyme sites, multiple cloning
sites, other coding segment~, and the like, such that their overall length may vary
considerably. It is therefore col-lelll~ ted that a nucleic acid fra~mPnt of almost any
length may be employed, with the total length preferably being limited by the ease of
p~ tinn and use in the intended recombinant DNA protocol. For example, nucleic
acid fr~gmPnt~ may be yr~par~d which include a short stretch complPmPnt~ry to SEQ
ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:47 and SEQ ID NO:48, such as about 10
nucleotides, and which are up to 10,000 or 5,000 base pairs in length, with segmPnt~
of 500 being yr~felled in most cases. DNA segment~ with total lengths of about 1,000,
500, 200, 100 and about 50 base pairs in length are also conl~l"~ t~ to be useful.
It will also be understood that this invention is not limited to the particular
nucleic acid and amino acid sequences of SEQ ID NO:l, SEQ ID NO:2, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:47,
SEQ ID NO:48, and SEQ ID NO:49. Recombinant vectors and isolated DNA
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21
segmPnt~ may th~refof~ variously include the dirrel~nLially expressed coding regions
or the BRCAI coding regions thPm~Plves, coding regions bearing selected alterations
or m~ifiç~tion~ in the basic coding region, or they may encode larger polypeptides
- which nevertheless include diLre~ tially eA~l~ssed-coding regions and the BRCAl
S coding regions or may encode biologically function~l equivalent proteins or peptides
which have variant amino acids sequences.
The DNA segmP,nt~ of the present invention encompass biologically functional
equivalent differentially ~;A~r~ i,sed proteins and peptides biologically functional
equivalent proteins of BRCAl. Such sequences may arise as a consequence of codonredlln-lancy and functional equivalency which are known to occur naturally within
nucleic acid sequences and the proteins thus encoded. ~ltern~tively, functionally
equivalent proteins or peptides may be created via the application of recombinant DNA
technology, in which changes in the protein structure may be PnginPPred, based on
considerations of the pr~,~ Lies of the amino acids being exchanged. Changes dç~ignPA
by man may be introduced through the application of site-directed mutagenesis
techniques, e.g., to introduce improvements to the antigenicity of the protein or to test
site-dilecLed 1ll~ or others in order to PY~mine carcinogenic activity of the
differentially t;A~l~;ssed marker genes at the molecular level.
If desired, one may also plc~ fusion proteins and peptides, e.g., where the
dirL~ ally ~;A~r~ marker gene coding regions are aligned within the same
eA~lts~ion unit with other proteins or peptides having desired functions, such as for
purifir~tinn or immun~etP~ti~ n purposes (e.g., proteins which may be purified by
affinity cl~vllla~ogl~hy and enzyme label coding regions, respectively).
Recombinant vectors form iln~ol~lt further aspects of the present invention.
Particularly useful vectors are con~e~pl~tp~ to be those vectors in which the coding
portion of the DNA segmPnt is positionP~ under the control of a promoter. The
promoter may be in the form of the promoter which is naturally associated with aRIBRED gene, e.g., in human cells, as may be obtained by i~ol~tinp the S' non-coding
sequences located UL~SL~ ll of the coding segment or exon, for example, using
recombinant cloning and/or PCR technology, in connection with the compositions
disclosed herein.
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In other emb~1imentc, it is co~ ,npl~te-l that certain advantages will be gainedby positioning the coding DNA segment under the control of a recombinant, or
heterologous, promoter. As used herein, a recombinant or heterologous promoter is
inten-ied to refer to a promoter that is not normally ~ccoci~t~d with a dirrere, lially
eAyressed marker gene or the BRCAl gene in its natural environmellt Such promoters
may include MMTV promoters norm~lly associated with other genes, and/or promoters
isolated from any other b~-teri~l, viral, eukaryotic, or m~mm~ n cell. Naturally, it
will be important to employ a promoter that effectively directs the t;Ayrt;ssion of the
DNA segm~nt in the cell type chosen for ~Ayression. The use of promoter and celltype combinations for protein eAyl~ssion is generally known to those of skill in the art
of molecular biology, for example, see Sambrook et al. (1989). The promoters
employed may be conctih-tive~ or inducible, and can be used under the apLpfoy~iate
conditions to direct high level eA~ression of the introduced DNA segm~-nt7 such as is
advantageous in the large-scale production of recombinant proteins or peptides.
Appropriate promoter systems con~r.,.~ ted for use in high-level expression include,
but are not limited to ayy~pliate b~teri~l promoters.
As mentioned above, in connection with expression embo~im.ontc to ylcpare
recombinant differentially eAyiessed marker gene encoded proteins and peptides, it is
C()~ lpl~t~ that longer DNA segm~ntc will most often be used, with DNA segments
encoding the entire dirrer~;l-tially ~Aylessed protein or subunit being most yler~rr~d.
However, it will be appreciated that the use of shorter DNA segmPntc to direct the
~Ayres~ion of diffe~ tially ~Ay~essed peptides or epitopic core regions, such as may
be used to generate anti-marker protein antibodies, also falls within the scope of the
invention (EIarlow et al, 1988).
DNA se~m~ontc which encode peptide antigens from about 15 to about 50
amino acids in length, or more preferably, from about 15 to about 30 amino acids in
length are contempl~ted to be particularly useful. The C terminus of proteins provide
an excellent region for peptide antigen recogition (Harlow et al, 1988). DNA segments
encoding peptides will generally have a minimllm coding length in the order of about
45 to about 147, or to about 90 nucleotides. DNA ~gm~ntc encoding partial lengthpeptides may have a minimllm coding length in the order of about 50 nucleotides for
-
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23
a polypeptide in accoldallce with seq id no:3, or about 264 nucleotides for a
polypeptide in accoldallce with SEQID NO:l.
In ~ddition to their use in directing the expression of the dirre~lltially ~A~essed
- marker proteins, the nucleic acid sequences disclosed herein also have a variety of other
uses. For example, they also have utility as probes or primers in nucleic acid
hybridi7~tion embo limPnt.e. As such, it is contelllplated that oligonucleotide fr~gmPnte
corresponding to the sequences of SEQID NO:l, SEQID NO:2, SEQID NO:3, SEQ
ID NO:4, SEQID NO:5, SEQID NO:6, and SEQID NO:7 for stretches of be~w~
about 10 to 15 nucleotides and about 20 to 30 nucleotides will find particular utility.
Longer complPn~ .. y sequences, e.g., those of about 40, 50, 100, 200, 500, 1000,
and even up to full length sequences of about 2,000 nucleotides in length, will also be
of use in certain embo.1imPnte.
The ability of such nucleic acid probes to specifically hybridize to dirrer~nlially
~ressed marker gene sequences will enable them to be of use in detecting the
presence of complemPnt~ry sequences in a given sample. However, other uses are
envisioned, inclu~ling the use of the sequence information for the pr~al~ion of mutant
species primers, or primers for use in ple~ g other genetic constructif-ne.
Nucleic acid molecules having stretches of 20, 30, 50, or even of 500
nucleotides or so, comrlem~pnt~ry to SEQID NO:l, SEQID NO:2, SEQID NO:3,
SEQID NO:4, SEQID NO:5, SEQID NO:6, and SEQID NO:7 are particularly
col-~P.-.pl~tP~ as hybri(li7~tion probes for use in, e.g., Southern and Northern blotting.
This would allow dirrelc~lllially t;~r~ssed structural or regulatory genes to be analyzed,
both in p~tiPnte and sample tissue from pre-invasive and invasive breast tissue. The
total size of fr~gmPnt as well as the size of the complemP-nt~ry stretch(es), will
ultim~tely depend on the intPn~le~ use or application of the particular nucleic acid
seg.n.~l-t Smaller fr~gmPnte will generally find use in hybri~li7~tion embo(1im~Pnte,
wherein the length of the complPmPnt~ry region may be varied, such as between about
10 and about 100 nucleotides, but larger complPmPnt~ry stretches of up to about 300
nucleotides may be used, according to the length complementary sequences one wishes
to detect.
CA 022l0396 l997-07-l4
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24
Nucleic Acid Hyb.;~l;,~;., =
The use of a hybritli7~tiQn probe of about 10 nucleotides in length allows the
formation of a duplex molecule that is both stable and selective. Molecules having
comrl~ sequences over stretches greater than 10 bases in length are generally
S p~erelled, though, in order to increase stability and selectivity of the hybrid, and
thereby improve the quality and degree of specific hybrid molecules obtained. One will
generally prefer to design nucleic acid molecules having gene-complemPnt~ry stretches
of 15 to 20 nucleotides, or even longer where desired.
Hybri-1i7~tion probes may be selected from any portion of any of the sequences
disclosed herein. All that is r~uired is to review the sequences set forth in SEQ ID
NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,
and SEQ ID NO:7 and to select any continuous portion of one of the sequences, from
about 10 nucleotides in length up to and incl~l-ling the full length sequence, that one
wishes to utilise as a probe or primer. The choice of probe and primer sequences may
be governed by various factors, such as, by way of ~Px~mple only, one may wish to
employ primPr~ from towards the termini of the total sequence, or from the ends of the
functional domain-encoding sequences, in order to amplify further DNA; one may
employ probes corresponding to the entire DNA, or to the 5' region, to clone marker-
type genes from other species or to clone further marker-like or homologous genes
from any species inclu(1ing human; and one may employ randomly SPlPctP~d~ wild-type
and mutant probes or primers with sequences centered around the RibRed M2 subunit
encoding sequence to screen DNA samples for differentially ~ ressed levels of
RibRed, such as to identify human subjects which may be t;~ressing dirr~el,tial levels
of RibRed and thus may be susceptible to breast cancer.
The process of selPcting and ~.c;pa~ing a nucleic acid segment which includes
a sequence from within SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7 may ~llP.~ ively be described as
~prep~.;ng a nucleic acid fr~gmPnt". Of course, fr~gmPnt~ may also be obtained by
other techniques such as, e.g., by mP~h~nic~l ~h~ring or by restriction enzyme
rligestion. Small nucleic acid segmPnt.~ or fr~gmPnt~ may be readily pLepaled by, for
example, directly synthPsi7ing the fragment by chemical means, as is commonly
CA 02210396 1997-07-14
WO 95/19369 . PCT/US95/00608
practiced using an duLollldlPd oligonucleotide synthP~i7Pr. Also, fr~gmPnt~ may be
obtained by application of nucleic acid reproduction techn~logy, such as the PCRtechnology of U.S. Patent 4,603,102 (incoll,o~aled herein by reference), by introducing
ectpd sequences into recombin~nt vectors for recomhin~nt production, and by other
S recomhin~nt DNA techniques generally known to those of skill in the art of mol~P~ul~r
biology.
Accordingly, the nuclooti-lP sequences of the invention may be used for their
ability to selectively form duplex molecules with complçrnPnt~ry stretches of
dirre~ntially ~A~ssed marker genes or cDNAs. Depending on the application
envi~ionPd, one will desire to employ varying con-lition~ of hybritli7~tinn to achieve
varying degrees of selectivity of probe towards target sequence. For applications
l~uiling high selectivity, one will typically desire to employ relatively stringent
conditions to form the hybrids, e.g., one will select relatively low salt and\or high
lelll~ alule conditions, such as provided by 0.02M-O. lSM NaCl at ~,llpe dlu-es of
50~C to 70~C. Such selective conditions tolerate little, if any, mi~m~t~h between the
probe and the template or target strand, and would be particularly suitable for i~l~ting
specific dirre.e,llially C;A~ essed marker genes.
Of course, for some appli~tion~, for PY~mple, where one desires to p~ e
lllul~lL~, employing a mutant primer strand hybridi_ed to an underlying template or
where one seeks to isolate marker gene sequences from related species, functional
equivalents, or the like, less stringent hybritli7~tinn conditions will typically be needed
in order to allow form~tion of the heteroduplex. In these circum~t~nr~s, one maydesire to employ conditions such as 0.15M-0.9M salt, at lelll~ldlul~s r~nging from
20~C to 55~C. Cross-hybri~li7ing species can thereby be readily i~entifiP~l as positively
hybril1i7.ing signals with respect to control hybri-li7~tiQns. In any case, it is generally
a~pl~ialed that conditions can be rendered more stringent by the ~ ition of increasing
amounts of form~mide, which serves to destabilize the hybrid duplex in the same
manner as increased lelll~ldlu-~. Thus, hybril1i77/tion conditions can be readily
manipulated, and thus will generally be a method of choice depen-ling on the desired
results.
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26
In certain embolimPnt~, it will be advantageous to employ nucleic acid
sequences of the present invention in combination with an a~r~pliate means, such as
a label, for deler",inillg hybridi7~tion. A wide variety of a~liate in-lic~tor means
are known in the art, inrlll-ling fluo,c;sce"t, r~io~rtive, enzymatic or other lig~n-ls,
such as avidin/biotin, which are capable of giving a detect~ble signal. In ~ler~rled
emb~i~ nl~, one will likely desire to employ a fluorescent label or an enzyme tag,
such as urease, ~lk~linP phnsph~t~e or peroxid~e, instead of r~(lio~ctive or other
environmPnt~l undesirable reagents. In the case of enzyme tags, colorimptric indi~tor
substrates are known which can be employed to provide a means visible to the human
eye or s~ecLIo~)hotomPtric~lly, to identify specific hybridization with complemPnt~ry
nucleic acid-co~";-ining MmrlPs
In general, it is envisioned that the hybri~i7~tinn probes described herein willbe useful both as reagents in solution hybri~i7~tion as well as in embo(1imPnt~
employing a solid phase. In emborlimPnt~ involving a solid phase, the test DNA (or
RNA) is adsorbed or otherwise affixed to a selected matrix or s--rf~ee. This fixed,
single-stranded nucleic acid is then subjected to specific hybri~i7~tion with selected
probes under desired contlition~. The SPlP~t~pd conditions will depend on the particular
circum~t~nces based on the particular criteria required (clepentling, for example, on the
G+C con~ , type of t~rget nucleic acid, source of nucleic acid, size of hybricli7~tion
probe, etc.). Following washing of the hybridized surface so as to remove
non~pecific~lly bound probe molPcules" spPc-ific hybri~i7~tion is detectPd, or even
qll~ntifiPA7 by means of the label. (Sambrook et al, 1989).
In a ~rerelled embodiment of the mPth~l, certain prelimin~ry procedures are
nPce~ry to pl~e the Mmple tissue and the probes before the d~Pt~P~tion of dirr~l~;"tial
t;A~ression of marker genes in abnormal tissue as colllp~cd to that in normal tissue can
be accompli~hPd
SAMPLE PREPARATION
RNA pllrific~tion
RNA was isolated from frozen tissue samples by mincing of micro li~ect~d
fro~n tissue fr~gmPnt.~ with a ra_or blade and then adding 800 microliter of 5.6M
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27
g~l~nitlinillm to increase mixing, followed by a 30 second microcentrifuge cenllifugation
at 14,000 rpm to remove particulate matter. The ~u~, Il~I;.nt was then removed and
the viscosity was reduced by multiple aspirations through a 22 gauge needle and then
- 200 ul of chloloform was added and the sample was ins~ub~d on ice for 15 ",;n~es
(during this time the sample was vortexed mllltiI)le times). Following incub~tion with
chloroform, the sample was ~n~iruged for 15 mimltes at 14,000 rpm and the aqueous
layer was removed and ethanol p~ i~led. This PYtr~rtion method produces RNA
which is l~,;.,-;1-;ly derived from cells of epithelial origin. In order to obtain RNA
~mples which ~.esu",ably in~ludes RNA derived from these stromal cells; the
particulate m~tPri~l (,~;.. ~i,.il.g in the pellet from the 30 second cer,llifugation) was
homogenized with a t~ emi7pr~ washed with PBS, treated with collagenase at 37~C
for 30 ."inl.les, soni~tP~, PYtr~rtecl with phenol/chlo,oro~lll and ethanol p~eci~ led.
cDNA libraries were constructed in lambda phage using polyA-selected mRNA
from the following samples; cultured human breast epithelial cells, tissue from three
reduction m~mmoplasty p~tiPnt~, tissue from three DCIS p~tient~, and tissue from one
DCIS patient (patient #10) that showed a focus of microinvasion ~ ~nt to an area of
DCIS. Mll1tiple punches were needed to obtain sllfficiPnt RNA for polyA sPlPction and
library construction. 200 ug of total RNA was obtained by pooling 20 punches from
normal breast tissue (reduction m~mmoplasty ~mples) and 5-8 punches from DCIS
le~ion~ su~lably reflPcting the greater cPlllll~rity of the DCIS ~mplPs cDNA
libraries were constructed by first and second strand cDNA synthesis followed by the
~ itiQn of direction~l synthetic linkers (ZAP-cDNA Synthesis Kit, Stratagene, LaJolla, California). The Xho I-Eco Rl linkered cDNA was then ligated into lambda
arms, packaged with pac~ging extracts, and then used to infect XL1-blue bacteriareslllting in cDNA libraries.
PROBE PREPARATION
The collagen m probe employed for nuclease plolecLion assays was constructed
by subcloning the 208 bp Hinc II-Pst I fr~gmPnt from the 3' ~mt~n~l~ted region of the
human type m procollagen gene into pGem4Z. This region of the human procollagen
m gene was obtained by PCR amplific~tion of published sequence (Ala-Kokko et al,1989) followed by restriction with Hinc II and Pst I. For a control probe to assure
CA 02210396 1997-07-14
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equal loading and recovery of RNA, we used a T7 polymerase-gPner~tPd probe for
human glycerAlrlehyde phosphate dehydrogenase (GADP) which protects a 140 bp SacI-Xba I frAgmPnt; (a generous gift from Janice Nigro, Vanderbilt University). Probe
DCIS-l was genPrAtP~l by linPAri7ing the rescued plasmid with Pvu II, which should
S generate a 200 bp ~fole~d frAgm~Pnt RNase plote~;lion assays were pelrulll-ed with
1 ug of un~PlP~te~ RNA and the above-cited probes using the methods we have reported
previously (Holt, 1993).
Dirrt;lelltial Display-based cloning of cDNAs:
Rescued cDNA library samples were use~d as templAtPs for low stringency PCR
with the either a pair of 25 bp primers or an anchored 14 bp primer paired with a
random 25 bp primer. Random 25 bp primers were generated by a co~ u~el-based
algo~ n, (Jotte and Holt, unpublished). SAmples were denatured for two ~ lles at95~C followed by 40 cycles, each cycle con~isting of denalul~lion for 1 minute at
94~C., AnnPAling for 2 ",i~-u~es at 25~C., and extension for 1 minute at 72~C. The
samples were then run on an 6% non-denAtllring polyacrylamide gel, which was dried
and autoradiogrAph~d. Specific bands were excised then reamplified with the sameprimers used for their genPrAtion. Spe~ifi~ity was conrlr",ed on 6% polyacrylamide
gel, and ~Ampl~Ps were purified by ethanol plec;~ nn of the remAin~er of the PCRreaction. FrApmPnt~ were then individually cloned into Srff cut vectors by standard
methods using PCR-ScriptTMSK(+) Cloning Kit (Stratagene, LaJolla, California) and
then sequenced.
E~AMPLE 1
Studies showin~ Increased Risk of Breast Cancer
in Patients with DCIS
Since the 1970's, studies of pre-invasive lesions associated with the development
of breast cancer have been undertaken in an aLl~l"pt to refine histologic and cytologic
criteria for the hyperplastic lesions analogous to those of the uterine cervix and colon.
Rel~ P of the availability of tissue from breast biopsies done many years previously,
cohorts of women who underwent breast biopsies 15 to 20 years ago, can be studied
to del~ ",in~ the risk for development of breast cancer attributable to specific lesions.
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29
Many concurrent studies ev~ ting lesions associated with cancer at time of cancer
diagnosis led the way in pointing out lesions of potential i~ e~l (Wellings et al, 1975).
Hopefully, these intPrmPAi~tP stages in cancer development will serve to providein.1i~tors of breast cancer developnlent sllfficiently precise to guide prevention and
intervention str~tp~gi-ps (Weed et al, 1990; Lippman et al, 1990). Such intPrmPAi~tP
elemPnt~ prior to the development of mPt~t~tic capable cancers also provide the
o~ luniLy to define the molecular biology of these Pl~mPnt~ Studies of the
development of pre-invasive breast disease have provided insight into difrelcllt types
of lesions with dirrelcllt implications for breast cancer risk and the process of
carcinogenesis (See Figure 1). Pre-invasive breast disease is herewith dPfinPA to be any
reproducibly defined condition which confers an elevated risk of breast cancer
appro~ehing double that of the general population (Kon~itowski et al, 1990). Thespecifically-definPA atypical hyperplasias and lobular carcinoma in situ confer relative
risks of four to ten times that of the general population. This risk is for carcinoma to
develop anywhere in either breast (Page et al, 1985; Page et al, 1991). The st~ti~tir~l
signific~nce of these observations have regularly been <.0001. Thus, absolute risk
figures of 10-20% likPlihood of developing into invasive carcinoma in 10 to 15 years
arise. DCIS is a very special elPmPnt in this story because the m~gnit~lde of risk is as
high as any other conr1ition noted (P~ .00005), but lc~ ;-hly, the developing
invasive cancer is in the same site in the same breast. This local lc~;ullcl~ce and
evolution to invasivel-ess marks these lesions as de~ ",;n~te preC;Ul~Ol;j of invasive
breast cancer (Betsill et al, 1978; Page et al, 1982). These figures are for the type of
DCIS which has become detectP~ very commonly since the advent of m~mmography,
the small and NCDCIS variety. It is likely that the comedo DCIS variety in~li~tps a
much greater risk, often plCSe~ )g as larger lesions, and treated regularly by
m~lec~ ly in the past 50 years making follow-up studies impossible (Figure 1).
The precision of histopathologic tli~gnosi~ in this area as noted in Table I (shown
in Figure 1) was most convincingly conf..llled in a large, prospective study (London
et al, 1991). There has also been a recent review of the reproducibility of the
~ignmPnt of ~ gnosi~ by a panel of pathologists (Schnitt et al, 1992). The precision
has been fostered by combining histologic pattern criteria with cytologic and extent of
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lesion criteri~ Classic surgical pathology criteria were predomin~ntly derived from
histologic pattern only. A further point of relevance to the il"pol~lce of thesehistopathologically defined lesions of pre-m~lign~ncy in the breast is the relationship
to f~mili~lity. A family history of breast cancer in a first degree relatives confers about
a doubling of breast cancer risk. However, women with the atypical hyperplasias at
biopsy and a family history of breast cancer are at 9-10 times the risk of developing
invasive breast cancer as the general population (Dupont et al, 1985; Dupont et al,
1989).
Careful con~idPr~tiQn of all of the above-mentioned epidemiologic data has led
to the following model for progression from gener~li7pA pre-m~lign~nt lesions todelellllinallt lesions to invasive cancer. Figure 2 shows this model for the induction and
progression of pre-invasive breast disease based on study of the Vanderbilt çohort
(Dupont et al, 1985) of more than 10,000 breast biopsies (follow-up rate 85%; median
time of 17 years; 135 women developed breast cançer).
EXAMPLE 2
Identifiçation of Penes whiçh are dirre~ Lially eA~)lessed in DCIS
Construction of cDNA libraries from DCIS lesions
~ order to study dirrer~ntial gene ~ .ession in DCIS, we cnllecte~ cases of
NCDCIS. The diagnosis of DCIS is made on histomorphologic grounds based on
arçhitP~lral, cytologic, and ocç~ion~lly extent 5rit~ri~ NCDCIS lacks comedo
features and çonsists of microsçopic intr~ ct~l lesions which fill and extend the duct,
contain rigid intPrn~l ~çll;~ecl~..e, and often have hy~lchrolnalic and monomorphic
nuclei.
Study of non-çomedo DCIS for dirr~ lial marker gene ~ression inllir~tes the
diagnostic utility of co...p~ on of marker gene t;A~ression in these tissues. Although
the morbidity and mortality of breast cancer clearly results from invasion and
mPt~t~ , the development of breast cançer is clearly ~ignific~nt in its early stages for
two basic reasons:
1) The molecular changes will ~ mably be simpler in early lesions than
in later lesions which may have acquired numerous mutations or "hits";
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and
2) Successful prevention st~tegi~s may require ~tt~.king cancer before it
develops the capacity to invade or mPt~t~i7~.
Y Non-comedo DCIS is the earliest dele~ ant lesion which recurs locally as
invasive cancer. Although comedo DCIS may be te~hnic~11y easier to study becausethe tumors are larger, its agglessi~eness and the presence of numerous genetic
alterations (such as pS3 and erbB2) suggest that it may have advanced beyond theearliest stages of carcinogenesis.
The commercial utility of a method for prevention of cancer is clear. In order
to study dirr~lential gene ~L~ssion in DCIS, breast tissue with e~tensive microscopic
non-comedo DCIS was i~l~ntifi~ and banked in a frozen state. cDNA libraries wereconstructed from mRNA i.~o1~t~1 from frozen sections of DCIS lesions. Tissue ~mp1es
from p~ti~ont~ with m~mmographic results con~i~tent with DCIS were cryostat frozen
and a definitive ~ gnosi.~ was made by the histopathologic criteria which we have
described (Jensen et al, SulJll~ilLed for publication; Holt et al, In press).
Control mRNA was obtained from frozen tissue samples obtained from reduction
m~mmoplasties and from cultured human breast epitheli~1 cells. Rec~-1se non-comedo
DCIS is a microsco~ic lesion, we had to microlocalize regions of DCIS in biopsy
samples. To accomplish this we plepa,~;d frozen sections in which we located regions
of DCIS and then employed a 2 mm punch to obtain an abnGl,llal tissue sample only
from those regions that col1ti.in~d DCIS. This selective ha.~t;s~ g was accomp1i~h~1
by carefully ~1igning the frozen section slide with the frozen tissue block and
identirying areas of in~l~sl. The harvest of the a~prop iate area was then confirm~l
with a repeat frozen section. A similar approach was used to isolate mRNA from
lobules of normal breast in samples coll~te~l from a reduction m~mmoplasty. Prior
studies have shown that breast lobules are a~roxim~te1y 2.5 mm in ~ mtott r~ thus the
2 mm punch provided a well-tailored ~Y~i~ion. This microlocation and collection step,
in which abnormal tissue ~mp1es are co11e~t~d from an i~ol~t~hle tissue structure, was
pei~lllled with eYtreme care and was absolutely crucial to the success of these studies.
Con~ tion by normal breast epithelial cells or by breast stromal cells would clearly
negatively skew the dirrerel,tial screening approach. If the punch biopsy did not cleanly
CA 02210396 1997-07-14
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excise DCIS without col,t;1",~ tion by other cell types or tissues then the sample was
not used for mRNA isolation aensen et al, Submitted for publication). Figure 3
contains color photos of DCIS (abnormal) tissue, before (upper left panel) and after
excisional punch biopsy (upper right panel). The lower panels show tissue samples of
S normal breast tissue (lower left panel), and invasive breast cancer (lower right panel).
Following microlocation punch harvesting of the frozen tissue, RNA was isolated,purified, and employed to construct cDNA libraries. RNA was i~ol~tPA following
mincing of tissue in 5.6M g~lAni~linium isothiocyanate and 40% phenol, centrifugation
to remove particulate matter, viscosity reduction by repe~tP~ aspiration through a 22
gauge needle, chlorofolm extraction and ethanol precipitation. In most samples there
was particulate matter resistant to gll~ni~linillm-phenol PYtrActinn that was white in color
and fibrous in ap~A,i1~-ce and was presumPcl to r~resent breast stroma. This stromal
mAtPri~l was sparse in DCIS s~mples but abundant in samples obtained from normalbreast tissue derived from reduction mAmmoplasties. The stromal mAtPriAl was minced
with a ti.~uemi7PS~ washed with PBS, treated with collagenase at 37~C for 30 lllinules,
sonir~tPA extracted with phenol/chloroform and ethanol precipitated. 200 ug of total
RNA was obtained by pooling 20 punches from normal breast tissue (reduction
mammoplasty samples) and 5-8 punches from DCIS lesions, presumably reflecting the
greater cellularity of the DCIS samples. All libraries had greater than 50% inserts and
cont~ined bt;lween 2 X 106 and 7 X 107 phage recombinants with an average insert size
varying belween 500 and 1000 base pairs.
EXAMPLE 3
Development of an extraction method which produces breast epithelial RNA
It was neces~ry that tissue samples not be contAminAtPd by non-epithelial stromal
cells. Such contAminAtion would complicate efforts to conl~a,c; gene expression
between samples. In order to test the extent of stromal co~ "~ tiQn of the mRNA
samples, we det~ ed the level of expression of collagen III mRNA by an RNase
prole;Lion assay. RNase protection assays were employed in these and subsequent
studies because it is a 4uA~ ti~le method and can be ~l~olllled on small amounts of
lln~PlPcte i RNA. Collagen m mRNA was identified in the prçsumed stromal fraction
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WO 95/19369 PCT/US95/00608
of the normal breast tissue and to a lesser extent in the microinvasive breast cancer
sample, but no t;~ssion of collagen m was detPcted in the DCIS ~mrlPs which weresubsequently employed for cDNA library construction. Figure 4 COlllp~heS t;A~l~ssion
in NL 2 and #lOCA with other patient samples and NLl to ~elP~ inP collagen m
S ~A~,ess-on.
Expression of Collagen III mRNA in tissue mRNA ~mrles was analyzed by
RNase protection assay by meth~$ we have reported previously (Holt, 1993). One ~g
of mRNA was hybridized with two labeled RNA probes: a T7 polymerase-genç~tçd
probe for human glyceraldehyde phosphate dehydrogenase (GADP) which protects a
140 bp Sac I-Xba I fr~gmPnt and a T7 polymerase-gPnPr~ted probe which pn~ a
208 bp Hinc II-Pst I fragment from the 3' untr~n~1~ted region of the human type m
procollagen gene (Coll III) obtained by PCR subcloning of the published sequence (Ala-
Kokko et al, 1991). RNA ~mples were labeled as follows: NLl is RNA from culturedhuman breast epithelial cells (Hammond et al, 1984), NL2 is RNA from normal breast
tissue, NL3 is RNA derived from the fibrous stromal fraction of breast tissue asdescribed (Jensen et al, Submitted for publi~tion), NL4 is another sample from normal
breast tissue. This is described in greater detail on page 30 of this patent appli~-~tion
#12,#8,#4,#6, and #10 are from patient ~mI)lPs with DCIS. Sample #lOCA is RNA
obtained from the small focus of microinvasion shown in Figure 3. Con is a control
sample using ~RNA.
E2~AMPLE 4
Screening of cDNA libraries
Following succP-~ful testing which demon~tr~t~ that stromal cnnl~",in~lion was
not a problem, cDNA libraries were constructed in lambda phage using polyA-selected
mRNA from the following ~mples- cultured human breast epithelial cells, tissue from
three reducti~ n m~mmoplasty p~tiPnt.C, tissue from three DCIS p~tiPnt.~, and tissue from
one DCIS patient (patient #10) that showed a small focus of invasion ~ nt to an
area of DCIS. Mllltirle punches were needed to obtain s~-fficient RNA for polyA
selecticn and library construction. Selective h~n-lling of tissue was accompli~h~.
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34
Comr~ri~on of gene ~Apression belw~n ~mrlPs was p~lro~llled by either
dirr~relltial screening or a modifir~tion of dirr~ ial display (Liang et al, 1992a; Liang
et al, 1992b; Saiki et al, 1988; Melton et al, 1984). Plasmid DNA was ~l~;p~ed from
the cDNA libraries following helper phage rescue and screened by two independentmPthods. Figure 5 below shows the results of differential display co,.,~ g cDNAsof several patient DCIS samples with cDNA obtained from normal breast epitheli~lcells and an early invasive cancer. Although few genes shown in this Figure are
dirrerelllially expressed in the majority of ~mrles with DCIS, the heterogeneity of gene
expression in patient samples is seen.
O The dirrelelltial display method (Liang et al, 1992a and 1992b) allows ~imlllt~neous
co",~ on of multiple tissue ~mples. Initial studies using this method (reverse
tr~n~çriI~tase followed by PCR) were un~ti~fartc)ry because of ullw~llt;d ~mI)lifir~tinn
of cont~min~ting DNA in tissue samples and the small size of many of the fr~gmPnt~
idPntified by display. To circumvent some of these problems, we have al~e,ll~led to
combine the advantages of cDNA library seleel-i,-g with the advantages of dirrt;lt;l~lial
display by:
1) Constructing cDNA libraries from the tissue mRNA ~mrles;
2) Pelroll~ g dirrert;~lial display on the plasmid DNA prepared from the
cDNA libraries;
--0 3) Subcloning the fr~gmPnt~ idçntifiçd by dirr~ lial display;
4) Using the subcloned fr~gmPnt as a probe to clone the cDNA from the
a~pl~pliate library.
F., ~ 5
Identification of a ~ene (RibRed) which is dirrerentially e~l"essed in multiple
-5 NCDCIS cases
Employing these methods, 10 ~liLr~lcllLially t;Apressed clones were i~lPntified and
the seven that showed the g.eate~l dirreLence in e~lc;ssion between mllltirlP s~mrlP.s
were further char~t~-tP-ri7~ by DNA seqllPncing. Comr~ri~on of the sequenced clones
with Ge-nR~nk demon~tr~t~1 that six of the clones are appa.elllly unique sequences
O (although further DNA sequPncinp is nPce~ry); but that one of the clones (here
termed DCIS-l and described in Sequence Listing No. 1) showed 90% homology to the
CA 02210396 1997-07-14
WO 95/19369 . PCT/US95/00608
previously cloned h~m~tçr gene encoding the M2 subunit of ribonucleotide re~uct~e
(Pavloff et al, 1992; Hurta et al, 1991; Hurta et al, 1991). Although human M2
ribonucleotide reduct~.~P has been cloned previously, comr~ri~on of the h~m~ter cDNA
- sequence with our clone and with the prior human clone intlie~te.s that DCIS-l is
S homologous to an ~ ely poly-adenylated form of the human ribonucleotide
reduct~e which has not been cloned previously. Figure 6 shows a co~ ~ison of thesequence b~lweell DCIS-l and the human and h~m.~ter genes.
Rec~llse of our concern that dirre ~nt p~tient~ may have dirr~c;nlial gene
t;A~ression which is idiosyncratic (or related to morphological differences in biopsy
appeal~lce) and not nPce.~rily related to the induction or progression of DCIS, we
~imlllt~neously analyzed gene ~Aplession in multiple DCIS samples co---pal~d to
multiple control ~mr~les. We constructed cDNA libraries from the following ~mples
1) Cultured HMEC epithPli~l cells;
2) ~eduction m~mmoplasty: 11 year old with virginal hyperplasia;
3) ~uction m~mmoplasty: 28 year old patient;
4) Reduction m~mmt)plasty: 35 year old patient;
5) DCIS patient #12;
6) DCIS patient #8;
7) DCIS patient #10;
8) DCIS patient #10 from an area of invasive cancer ~dj~r.e.nt to DCIS;
In ~ 1ition to the .~mrles we employed to construct cDNA libraries shown
above, we also obtained frozen tissue ~mples from 7 more DCIS p~tient~, 2 cellular
fibroadenoma samples, and ~mples of "usual hyperplasia" and atypical hyperplasia.
Rec~lse the DCIS clones were identifiel1 by cloning methods which include
selection and amplific~tion~ it was i~ t to confirm by n~lc~ e protection assaysthat the genes were dirrt cnlially ~A~lessed in the origin~l un~elP~tP~ m~mrlified RNA
samples (Figure 7).
This approach allowed i~lPntifir~tion of a human gene similar to the h~m~tPr RibRed
gene (coding for the M2 subunit) and 7 other human genes as genes which are
dirrelel.tially tA~l~;ssed in a majority of cases of DCIS in human breast tissue. The
table of dirr~lt;lltially ~Ayl~ssed genes lists the genes which have been i~lP.ntifiP~ as
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dirrclenlially cAplcssed genes in DCIS tissue samples as colllparcd to that in normal
tissue (Figure 9).
EXAMPLE 6
Methods for studyin~ ~otential use of dirrelenlially cA~r~ssed ~enes for tli~pnQstic
screening
One advantage of the differential display method is that it allows co.,~ - ;eon of
multiple tissue samples of pre-invasive or invasive breast cancer. For example, use of
this method has succe-eefully demonstrated that the M2 subunit ribonucleotide reduct;lee
gene is dirrcrcntially cA~lcssed in 4 out of 5 pre-invasive breast cancer tissue samples.
It is eignifir~nt that the M2 subunit is involved in the regulation of the ribonucleotide
re~luct~ gene and is found to be over-cAl)ressed in abnormal tissue samples.
Tdentifit~tion of dirrclc~ltially cAprcssed genes may lead to discovery of geneswhich are potentially useful for breast cancer screening. Of particular interest are
genes whose cA~ression is restricted to breast epithelial cells and whose gene products
are secreted. Screening for secreted proteins is possible by using the known
hydl~hobic sequences which encode leader se~quences as one primer for dirr~ ial
display. The i~f-ntific;ltion of secreted proteins which are specific for early breast pre-
m~lign~ncy (or even early invasive cancer) would provide an illlpol~lt tool for early
breast cancer scrccl~ing programs. If a dirrelc,ltially cApr~ssed gene has not been
cloned previously (or if details of its cAprcssion are unknown or uncertain) then
n~lclP~ee protection assays or Northern blots can be p~lrulllled on RNA pre~arcd from
tissue ~mplf-s from a variety of tissues to determine if eA~lcs~ion of this gene is
restricted to breast. If n~e~y cDNA libraries p.cpa~ed from other tissues can beadded to the differential display screen as a way to identify only those genes which are
cAI)~essed in early breast cancer and, in ~ltliti~n~ are only cAplcssed in breast tissue.
Once dirrelentially eA~lcsscd genes have been initially characteri~d for cAL~rcssion
in pre-m~lign~nt and m~ n~nt breast ~ ef-, antibodies to the protein products ofpotentially useful genes can be developed and employed for immunohietoçhf-mietry(Harlow et al, 1988). This will provide an ~ lition~l test to delclllline whether the
eAl,rcssion of this gene is restricted tû the breast. Subsequently, these antibodies will
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be used to detect the presence of this protein present in the blood of p~*ent.~ with pre-
invasive and/or invasive cancer. By assaying for serum protein levels in the same
p~*~nt~ who exhibited elevated ~Apression of the gene in their tissue .~mples it will be
- possible to d~ltlll~ine whether a gene product is being secreted into the blood.
s
EXAMPLE 7
D~l eased expression of BRCAl acc~ .tes growth and is obse~ ~ed during breast
cancer progression
Breast cancer occurs in hereditary and sporadic forms. Recently the BRCA 1
gene has been cloned and shown to be mllt~t~ in kindreds with hereditary breast and
ovarian cancer (Hall et al. 1990, Miki, Y. et al. 1994, Frie~m~n et al. 1994, Castilla
et al. 1994, Simard et al. 1994). Although 92% of f~milies with two or more cases of
early-onset breast cancer and two cases of ovarian cancer have germ-line mllt~*on~ in
BRCA 1 (Narod et al. in press), the gene has not been shown to be mllt~ted in any
truly sporadic case to date (Futreal et al. 1994). Despite the surprising paucity of
som~tic~lly acquired mutations in sporadic breast cancer, it is still a likely tumor
~u~ressor gene with a key role in breast epithelial cell biology. The BRCA 1 gene
encodes a protein of 1863 amino acids with a predicted zinc finger domain observed
in proteins which regulate gene tr~n~çriI)tion.
As an initial ch~r~cteri7~*nn of the regulation and function of the BRCA 1 gene,we analyzed and manipulated ~Ayles~ion of BRCA 1 mRNA levels. The results taken
together indicate that the BRCA 1 gene product is a negative regulator of m~mm~ry cell
proliferation which is e;Apressed at rlimini~h~d levels in sporadic breast cancer.
~;on of BRCAl mRNA during breast cancer ~ oCl~ion
As described above, microscopy-directed cloning has been employed to colllpa~e
gene t;Aylession in normal m~mm~ry epithPlillm, carcinoma in-situ, and invasive breast
cancer. This method produces predo,l~ ly epithelial mRNA with minim~l
cnnt;~ h-~ti-)n from stromal elemPnt~ and we used this approach to obtain mRNA from
normal neoplastic tissues from p~*Pnt~ without a family history of breast cancer.
Expression of BRCAl exon 24 in human breast tissue samples is shown in Fig. 1. The
legend of Fig. 1 is as follows.
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The following tissue ~mpl~-s were used for mRNA isolation: Normal tissue
,~mples NLl-cultured human breast epith~ l cells, NL2- Histologically normal breast
tissue from an 11 year old undergoing a reduction m~mmoplasty~ NL4- histologically
normal breast tissue from an 14 year old undergoing a reduction mammoplasty.
5Carcinoma-in-situ ~mplPs are #6, #8, #10, #12, #23 (comedo type), #41, #55; andinvasive cancer ~mples #lOCA (invasive cancer from the same patient with carcinoma-
in-situ), 36CA, lCA. All of these tissue .~mples were obtained from p~tit~nt~ who had
no family history of hereditary breast cancer and RNA p~ on was p~lro~ ed as
described above.
OPCR detecti~n of BRCAl exon 24 in cDNA libraries from the following tissue
~mples is described in Figure lOA. Lane 1: human genomic DNA, lane 2: NLl, lane
3: NL4, lane 4: $8, lane 5: #12, lane 6: #10, lane 7: #lOCA, lane 8: #41, lane 9: #23,
lane 10: 36CA, lane 11: lambda DNA. The arrow points to the expected 113 bp band.
Nuclease protection assays of micro li~ectecl mRNA from tissue samples are
5described in Fig. lOB. One ug of mRNA from each tissue sample was hybridized with
32P-labelled, T7 polymerase-genPr~ted RNA probes for BRCAl and human
glyceraldehyde-3-phosphate dehydr(~genase (GAPD) which produce expected pr~ ed
fr~gm~ont~ of 113 and 140 respectively as in~ ~tPA by the lines on the right. Data were
nl;l;ltP.d by phosphorim~ging. The hybrilli7ing intensity of each BRCAl band was~onorm~li7~d to its le~ ive GAPD band. The norm~li7~1 values of NLl, NL2, and
NL4 were intensity in each sample ,~lalive to 1. Sample 1 employs human leukocyte
mRNA; Samples 2-4 are NLl, NL2, and NL4; .~mples 5-9 are #6(2.8), 8(3.7),
10(2.8), 12 (5.9), and 55 (1.4); and 10-12 are #lOCA (0.07), 36CA (0.13), and lCA
(0.2).
~5Fig. 10 shows that BRCAl exon 24 mRNA is e~lessed at 5-10 fold higher
levels in normal ,.. ~.. ~. ~ tissue than in invasive breast cancer samples. Initial studies
showed ~lete~t~hle levels of BRCAl cDNA in a cDNA library ~r~a~ed from a tissue
sample with preinvasive carcinoma-in-situ but not in normal breast cancer invasive
breast cancer cDNA libraries (Figure lOA). Re~ lse this method is relatively
,oin.~en~itive we directly 4~l~nlil;-ted BRCAl mRNA by mlc1e~e ~ te~;lion assays in
RNA samples obtained by our microdissection method described above. These assays
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in~ te that ~AI)ression of BRCAl mRNA in micro-~ ectel normal ,.,~."...~.y
epithPli~l tissue (lanes 2-4, Figure lOB) is 5-15 fold higher than that in breast cancer
(lanes 10-12, Figure lOB). The highest levels of BRCAl are observed in ~mplPs from
- non-comedo ductal carcinoma-in-situ (lanes 5-9, Figure lOB), a prem~ n~nt breast
S lesion with a finite, but relatively low rate of progression to invasion (Betsill et at.,
1978, Page, D.L. et al., 1982, Page and Dupont, 1990).
Rec~ e these studies suggested that invasive breast cancer exhibited lower
mRNA levels than normal breast epithelial cells, we con~aled expression of paired
samples of normal breast and invasive cancer from the same patient (Figure llA;
O co"~art; lanes 2 and 3, 4 and 5, 6 and 7). The legend of Fig. 11 is as follows.
Nuclease plvleclion assays of RNA obtained from paired samples of invasive
breast cancer and histologically normal breast tissue are shown in Fig. 1 lA. Samples
in lanes 2 and 3 (first patient), 4 and 5 (second patient), 6 and 7 (third patient) are from
invasive cancer and normal breast tissue respectively. Lane 1 is NLl mRNA as
descAbed in legend to Fig. 10 and lane 8 is human leukocyte mRNA. Ratios of
BRCAl/GAPD for each ~mple: lane 1: 25.9, lane 2: 1.8, lane 3: 7.6, lane 4: 2.0,
lane 5: 12.4, lane 6: 0.7, lane 7: 6Ø The probes and methods are as described in Fig.
10 eAcept the GAPD probe was of lower specific activity to improve ~ n~ t;on.
Nuclease plotec!;on assays of RNA from a seAes of invasive breast cancer tissue
0 samples ~anes 2-9 colllp~ed with NLl (lane 1) and leukocyte mRNA (lane 10) are
shown in Fig. llB. Ratios of BRCAl/GAPD for each sample: lane 1: 19.1, lane 2:
0.3, lane 3: 1.8, lane 4: 1.6, lane 5: 0.2, lane 6: 0.3, lane 7: 1.9, lane 8: 0, lane 9:
0.6.
Although the samples were paired in Fig. l lA, they were not micro li~sected
~5 so this approach overestim~tes the relative eApression level of invasive ~mples because
they have a greater pel~nl~ge of epith~ l cells. RNA levels were four to eight fold
higher in ~mples deAved from normal breast than in samples deAved from invasive
breast cancer. We neAt analyzed t;A~Lession levels in 8 non-hereditary invasive cancer
samples (Figure llB: lanes 2-7). Although these samples showed some variability in
0 ~Ap~ession level, all had lower levels of BRCAl mRNA (de~ -lined by ratio of
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BRCAl to GAPD) than the primary breast epithPli~l cell line or the normal breast~mplPs shown in Figure llA.
Effects of BRCA1 gene inhibition on proliferative rate and gene expression
Having den tnetr~t~d that mRNA e~rc~ion levels of BRCAl are higher in
S normal .. ~,.. ~.y cells than in cancer cells, we used ~ntieenee methods to test the
hypothesis that BRCAl e~,cs~ion inhibits cell growth. Unmodified 18 base
deoxyribonucleotide complemP-nt~ry to the BRCAl tr~n~l~tiQn initi~tion site weresynthPei7Pd and added to cultures of primary m~mm~ry epithelial cells (Stampfer et al.
1980) or MCF-7 breast cancer cells (Soule and McGrath, 1980). Figure 12 is graphshowing growth rate of human plir,l~y .. ~.. ~.y epithpli~l cells (A), MCF-7 cells (B),
retinal pigmPntPA epithPli~l cells (C), cultured as described below. Points and bars
~rescllt the mean and the 95% confidPnre interval of triplicate counts of cells
incub~tPd with a single bolus of the in~lic~tP~ concentration of ~ntieçnee or control sense
deoxyribonuclPotide.
The morphologic a~ ce of the cell lines was not noticeably changed by
~irlition of ~nti.eenee oligonucleotide, but the proliferative rate was faster. Incubation
of cells with 40 uM anti-BRCAl oligonuclP~otide produced accelerated growth of both
normal (Figure 12A) and m~lign~nt m~mm~ry cells (Figure 12B), but did not affect the
growth of human retinal pigmPnted epithPli~l cells (Figure 12C). An intPrmPAi~te dose
of anti-BRCAl oligonucl~p~tide produced a less pronounced but signifir~nt increase in
cell growth rate. This was not a toxic effect of the oligonurlPotide since a control
"sense" oligomer with the same GC content did not increase the proliferation rate, and
because an addition of a 10 fold excess of sense oligomer to the anti-BRCAl oligomer
reversed the growth activation.
In order to critir~lly evaluate the function of BRCAl gene inhibition on growth
stim~ tinn and cell cycle progression it was nPcPe~y to identify a gene whose
cA~lCssion is cell cycle regulated in human ~ y cells. The gene encoding the M2
subunit of ribonucleotide re~uct~ee is amplified in conditions of nucleotide starvation
(Hurt~ and Wright 1992) and as shown above, exhibits elevated levels of expression in
prem~lign~nt breast diep~ee Re~-l$P ribonucleotide reductase conetitutes the rate
limiting step in DNA synth~eie, we reasoned that it might be cell cycle regulated in a
-- =
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synchronous growth model such as MCF-7 cells which can be growth arrested by
t~mrxifen and then restim~ t~l by estrogen (Aitken et al. 1985, Arteaga et al. 1989).
MCF-7 cells were growth arrested by t~moxif~n for 48 hours and then stimlll~tPA at
time zero (0) with luM estradiol (+E) or control vehicle (-E). Inhibition of DNAS synthesis by tamoxifen and in-luction of synthesis by estrogen were con~i""~l by
nuclear labelling studies with triti~t~l thymidine.
Fig. 13 panels A and B show that tr~n~cnption of the ribonucleotide re~uct~e
M2 gene is cell cycle regulated, inhibited by tamoxifen, and induceA by esllogell. Fig.
13A is a Northern blot of mRNA from synchroni~d MCF-7 cells. At the in-lic~ted
O time in hours, total cellular RNA was isolated and Northern blotting p~lro~",ed using
the 1.6 Kb Eco RI fr~m~nt from our cloned human ribonucleotide re~uct~ cDNA
described above. Two mRNA species of 1.6 and 3.4 Kb are observed in these studies.
Fig. 13B shows nuclear runon studies of synchronized MCF-7 cells were
S p~lÇol",ed by our published methods (Holt et al 1988) employing the 1.6 Kb fr~gment
of ribonucleotide re~luct~ described above (RR); the 1.8 Kb fragment of
Topoisomerase II (Topo) described in the Olsen et al. 1993); the 1.0 Kb cyclophilin
gene Clhompson et al. 1994) used as a con~ilulive control; and 18S ribosomal RNA(Thc,llll.son et al. 1994). Con l~senl~ cells which were grown for 48 hours but not
'0 treated with t~moxifen
~nti~n~ inhibition is a useful sll~dt~y for studying gene ~Aylession which is
dependent on t;A~l~ssion of the ~nti~pn~e target gene (Robinson-Benion and Holt, in
press, 1995), e.g. genes whose ~A~Lession is directly or indirectly depend~nt onBRCAl levels. Fig. 14 demonstrates that ~nti~en~ inhibition of BRCAl results in a
corresponding increased ~;A~lession of M2 ribonllcleotide reductase mRNA. A nucl~e
protection assay of mRNA derived from plilll~y ...~ .y epith~ l cells (lanes 1-4,
9-10) or MCF-7 cells (lanes 5-8, 11-12) cultured for 4 days with ~nti~Pn~e or control
oligonucleotide was p~lroll,led under the following conditions: no oligonucleotide (lanes
1 and 5); 40uM antiBRCAl (lanes 2,6,10,12); 4uM antiBRCAl (lanes 3 and 7); 40uM
~0 sense control (lanes 4,8,9,11). Probes for BRCAl and GAPD are as described for
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Figure 10, and the ribonucleotide reduct~e M2 probe (RR) detects the 200 bp probe
is described above.
Ribonucleotide reduch~e mRNA levels are highest in samples treated with 40
uM anti-BRCAl oligonucleotide for both primary ~ n~ y epithelial cells and for
S MCF-7 cells (Fig. 14). ~nti~Pn~e inhihition of BRCAl results in a 70-90% inhibition
of mRNA levels in anti-BRCAl treated cells co,n~ed with cells treated with the
"sense" control oligonucleotide (COll~ le lanes 9 and 10, Fig. 14). Note that MCF-7
cells have lower levels of BRCAl than the normal l~l~"~"~.y epithP~ cells (coll~afe
lanes 1 and 5, Fig. 14) anti-BRCA 1 since the ~nti~en~e inhibition may drop BRCAl
levels below a critical threshold which normally functions to inhibit growth.
Methodology
Tissue samples. Freshly obtained breast biopsy or reduction m~mmoplasty
sper,imPn~ were frozen and then RNA was obtained following the micro~ se~ti~ n
method described above. T~Psion~ were s~PlP~t~Pcl which were microloc~li7~d and
homogenous so that pure lesions could be obtained by 2 mm punches. Samples whichhad admixed normal epithpli~l~ carcinoma-in-situ, or invasive cancer were not used for
this study. Family history was obtained by chart review and/or interview to eYclllde
f~mili~l breast cancer cases.
Nuclease ~t~ Assays. PCR primers were derived from BRCAl
sequence in GenBank (Accession number U14680); forward 5'
CAATTGGGCAGATGTGT 3' and reverse 5' CTGGGGGATCTGGGGTATCA 3'
which amplify a 113 bp region from exon 24, corresponding to bases 5587 to 5699 of
the human BRCAl. This region was selPctçd because this exon has not been reported
to be dirr~ tially spliced unlike more 5' exons. The BRCAl probe was cloned by
subcloning this 113 bp band from normal human genomic DNA into PCRscriptSK and
screening for correct nri~nt~tion. One ug of mRNA from each tissue sample was
hybridized with 32P-labelled, 17 polymerase-generated RNA probes for BRCAl and
human glycp-r~ld~ohyde-3-phosphate dehydrogenase (GADP) which would produce
expected plolecled fr~gmlo,nt~ of 113 and 140 l~spe~ ely. The construction and use
of the GADP probe for RNA standardization has been described above. The probe for
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43
ribonucleotide reduct~e M2 mRNA is the same as above and detects a 200 bp
protected fr~gm~nt
A.~ ..ce oligonurl~ooti~ stn~ Unmodified deoxyribonucleotide were
- analyzed by gel electrophoresis and W shadowing and shown to be homogenous and
S of ap~r~iate size. These oligonucleotide were purified by multiple lyophili7~tic-n and
solubilized in buffered media as described (Holt et al. 1988). Sequence of the
unmodified antiBRCAl oligon~lcl~tide 5' AAGAGCAGATAAATCCAT 3' and the
complem~nt~ry sense oligonucleotide 5' ATGGATTTATCTGCTCTT 3' correspond to
the prelsllm~d tr~n~1~tiQn initi~tion site at bases 12-137 of the GenR~nk sequence. The
~nti~n~e oligonucleotide sequence was searched against Genb~nk and no ~i~nific~nt
homologies were identified to genes except BRCAl. Oligonucleotides were used
according to our publi~h~l mPth~l~ (Holt et al. 1988). Primary ",~"""~,~ epith~
cells were cultured in serum-free m~illm supplçmPnt~d with epiderm~l growth factor,
in~lllin, hydrocortisone, eth~nol~mine, phosphorylethanol~mine, and bovine ~iLuil~y
extract. MCF-7 cells were cultured in Minimllm F.~senti~l Medium _agle (Modified)
with Earle's salts and 2g/L sodium bicarbonate m supplçm~nted with 2mM L-
gl~ ",;l~, GMS-A (Gibco Cat. #680-1300AD), non.-~tonti~l amino acids, and 2.5%
fetal calf serum. Retinal pigmPnt~ perith~ l cells were cultured in DMEM and 10%calf serum.
Our results in~ te that the BRCA1 gene is t~)~t;Ssed at higher levels in normal
m~mm~ry cells than in breast cancer cells and that ~limini~hed e~,ession of BRCA1
increased the proliferative rate of breast cells. This correlates well with the recent
finding that p~ti~qnt~ with BRCAl gene-linked hereditary breast cancer have tumors that
grow more rapidly than co-,lp~dble sporadic tumors (Marcus, J. et al. 1994). Thedecreased mRNA levels which were observed in sporadic breast cancers are not a
consequence of dirrtl~ ial splicing of the gene since the RNAs were ~ nli~ ed with
probes from the 3' end of the mRNA which is not a region where dirrere .lial splicing
is reported to occur (~1~, Y. et al 1994). Invasive sporadic cancers have BRCAl
mRNA levels which vary from 0 (in one case) to 20% of the levels observed in normal
human m~mm~ry epith~ m.
~ =
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44
FY~mples 8 and 9 describe applic~tionc of the discovery of the function of the
BRCAl gene. FY~mple 8 describes a gene therapy method and eY~mple 9 describes
a drug scr~~ g method. The discovery of the (1imini~hP~l expression of the BRCAlmRNA in breast cancer using the micro ~ ection techniques of this invention provides
an illlpo~ sri~-ntific basis for these eY~mrlr.
FY~mple 8
Gene Therapy mlo.thod based on delP~,ui~ ion of the function of the BRCAl Gene
Viral vectors cQ~ g a DNA sequence that codes for a protein having an
amino acid sequence as ç~enti~lly set forth in SEQ ID NO:49 can be constructed using
iO techniques that are weU known in the art. This sequence inçl~des the BRCAl gene
product. Viral vectors conl~ g a DNA sequence es.s~ lly as set forth in SEQ ID
NO:47 (the BRCAl gene) can be also constructed using techniques that are well known
in the art. Retroviral vectors, adenoviral vectors, or adeno-~soci~tçd viral vectors are
all useful methods for delivering genes into breast cancer cells. An PY~llent c~n(li~te
~ S for use in breast cancer gene therapy is a Mo]on~y-based retroviral vector with a breast
selective MMTV promoter which we have reported previously (Wong et al). The viral
vector is constructed by cloning the DNA sequence esse.~ lly as set forth in SEQID:47 into a retroviral vector such as a breast selective vector. Most preferably, the
full-length (coding region) cDNA for BRCAl is cloned into the lc~ vil~l vector. The
~0 retroviral vector would then be tr~n~f~ted into virus producing cells in the following
manner: Viruses are p,ep~d by t~dnsrecling PA3 17 cells with retroviral vector DNAs
which were purified as described in Wong et al. Following transfection, the PA317
cells are split and then treated with G418 until individual clones can be il1çntified and
exp~nded. Each clone is then screened for its titer by analyzing its ability to transfer
-5 G418 re~i~t~nre (since the retroviral vector contains a Neol,lycin re~i~t~nce gene). The
clones which have the highest titer are then frozen in numerous aliquots and tested for
sterility, presence of replication-co.np~nt retrovirus, and presence of mycoplasm. The
methods generally employed for construction and production of l~LIovildl vectors have
been described in Muller, 1990.
Once high titer viral vector producing clones have been idçntififfl, then p~ti~.nt~
with breast cancer can be treated by the following protocol: Viral vector ~A~lessillg
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BRCAl is infused into either solid tumors or infused into m~lign~nt effusions as a
means for ~ltPring the growth of the tumor (since it is shown above that the BRCAl
gene product decreases the growth rate of breast cancer cells). Re~llsP viral vectors
can efficiP-ntly tr~n~ ce a high pclccnlage of cancer cells, the tumors would be growth
inhibited.
F.Y~mple 9
Method of Screening Compounds Capable of Activating Promoter Region of the
BRCAl Gene
The discovery of the function of the BRCAl gene provides a clear utility in thatinduction of cAplcssion of the gene and the resultin~ increase in level of protein
encoded by the gene in the breast cancer cell should slow the proliferation of the breast
cancer cells. Tnduction of cA~ression of the gene can be caused by ~mini~Pring acolllpoulld to a pa*ient that s*m~ tes the regulatory regions of this gene, such as the
promoter.
A method for scree~ g compounds that ac*ivate the promoter of the BRCAl
gene is de~ign~d in the following way. A promoter sequence is a DNA se~mPnt thatupregulates the cAl~ression of a gene. A sequence to-ssç~ lly as set forth in SEQ ID
NO:48 can be ligated into a suitable vector, such as a pl~mi(1, that contains a reporter
gene using standard recombinant DNA techniques of restric*on enzyme digests, liga*~on
of fr~gmPnt into vector, and tran~ro-"~;nn of b~Ct~ri~ SEQ ID NO:48 incllldes the
promoter sequence of the BRCAl gene. A lc~)ollcr gene is a gene that produces a
readily detect~hle product. F.Y~mples of a~L)r~pliate lCpO lcr genes which could be
employed for this purpose include Beta-galactosidase or the ~ hlnr~mph~ni~ol
acetyltransferase gene.
The BRCAl promoter/lcpol~cr gene combination can then be cloned into an
e~Apression vector or viral vector by standard recombinant DNA methods. Breast
cancer cells can then be transfected with the cA~lcs~ion vector co~ g the BRCAl
promoter/lc~ollel gene using standard tr~n~fection m~Shotl~ whichj we have reported
previously (Holt et al. PNAS 1986). A stable tran~rolll~ant with a~l)r~liale low level
cA~lcssion (breast cancer cells have low level BRCAl cA~lcssion as shown above) will
be i~P~tified and then characterized to demonstrate proper DNA integration and
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46
~A~lcs~ion. Methods of establishing and ch~tPri7ing stable transrc)~",~"l~ have been
described (Holt. MCB, 1994). Once an ~ropliate stable t~ansroll"~lt cell line isidentifiPcl, then we can plate the cell line in a manner than permits screening of
hundreds or thousands of drugs or biological agents (for eY~mple in mllltiple 96 well
S microtiter plates). Level of ~A~lcs~ion of the lcpolL~r gene can be ~ nt;lil~d and
agents which activate ~AL,ression are thus idPnt;fiff1 A positive result (i.e. in-luction
of the promoter region) results in increased levels of the re~.lel gene reslllting in
either an increase in color (Beta-galactosidase assay) or specific radioactivity(Chloramphenicol aceLylLldllsferase activity) through a reaction between the protein
encoded by the re~lLer gene and a co~ ound in the reaction mPAillm The compound
produced by the reaction between the lc~ltel gene protein and the compound in the
reaction m~Aillm is the cause of the increase in color or spe~ific r~io~tivity. These
coll,pounds can be called intli~t~r compounds in that their presence inflic~tes that the
drug or biologial agent activitated the BRCAl promoter. Methods for standardi_ing
and Lt;lrOll"illg Beta-galactosidase or chlor~mphenicol acetylt~nQfer~e assays have
been reported (Holt et. al. MCB 1994). This method would be useful for initial
scree~ g of agents which increase BRCAl ~lcssion. These agents could then be
tested in more rigorous assays of breast cancer growth such as nude mouse tumor
assays (Arteaga et al). This approach allows mass screel~ing of large numbers ofagents, sparing more rigorous animal tests for only promising colll~;>oullds which score
in the reporter gene assay described herein.
Thus, although there have been described particular embo iimPnt~ of the present
invention of a new and useful "Method for Detection and Tre~tn Pnt of Breast Cancer",
it is not intPn~e~ that such emb~limP-nt~ be construed as limit~ti~n.~ upon the scope of
this invention except as set forth in the following claims. It will be a~arellt to those
skilled in the art that many changes and mo lifiç~tions may be made without departing
from the invention in its broader aspects. For PY~mple, the above described techniques
may be used in t~he diagnosis of other ~ e~QP.s and detection of dirre~elltial genetic
~r~ssion from microscopically-directed tissue samples of pathologic tissue. The
production of a cDNA library produced as a result of the diLrel~elllial e~ression of
genes in pathologic tissue in col"p~ on to normal tissue provides the op~ u~ y for
CA 022l0396 l997-07-l4
WO 95/19369 PCT/US95/00608
47
further ~ gnostic capabilities. Further, although there have been described certain
experimPnt~l con~1itinn~ used in the p.~ell~d embotlimPnt it is not intPndPtl that such
conditions be construed as limit~tion~ upon the scope of this invention except as set
J forth in the claims.
S The following references are inclllded to provide details of scientific technology
herein inco~ ed by reference to the extent that they provide ~ lition~l inform~tion
for the purposes of in-li~ting the background of the invention or illustrating the state
of the art.
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52
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ADDITIONAL DESCRIPIION OF THE FIGURES
Figure 2: Model for prem~lign~nt contlition.c, highli~hting m~gnit~l-le of risk
for p.ogl~_ssion to clinical m~ n~ncy. Terms from human breast neoplasia are used:
no proliferative disease (No Pro), proliferative disease without alypia (PDWA), typical
hyperplasia (AH), ca.ci~ol,la in situ (CIS). As is proposal of tumor ~r~g~cssion each
stage is more likely to proceed to the next (dotted lines), but could also remain stable
(horizontal lines, probably fairly frequent), or directly proceed to develop a clone of
cells with m~lign~nt behavior (vertical lines, becoming more likely further to right.)
Figure 5: Dirrere,llial display of cDNAs obtained from patient tissue ~mrles
and controls. Rescued cDNA library c~mples were used as templates for low
stringency PCR with the primers 5'GATGAGTTCGTGTCCGTACAACTGG3' and 5'
GGTTATCGAAATCAGCCACAGCGCC3~; 40 cycles were ~; roll"ed at cl)n~litionc
described above. S~mr~lPs (See legend to Figure 4): Lane 1 - #12; Lanes 2 and 3:Se~i~t~ phage rescues of NL1 to show reproducibility of the assay; Lane 4 - #8; Lane
5 - #10; Lane 6 - #lOCA; Lane 7 - control from the rescued phage vector without
cDNA inserts. Arrows mark cDNAs which are uv~leA~l~ssed in DCIS versus normal.
Arrowheads mark cDNAs which are dirr~enlially ~A~lessed in the invasive cancer
(note this may reflect cont~min~tion from stromal cells). The bar marks a cDNA which
is t;A~ressed in normal breast cells at higher levels than in DCIS or invasive cancer.
Figure 7: EApression of DCIS-1 mRNA in tissue mRNA ~mpl~s analyzed by
RNase protection assay. Probes: GP~DH probe and DCIS-1 clone probe which was
generated by lin~ri7.ing the rescued plasmid with Pvu II and should generate a 200 bp
pl~ ed fr~gm~nt RNA samples were labeled as in the legend to Figure 4.
S1~511TU~E S~IEET (R~IL~ 2ff~
CA 02210396 1997-07-14
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SEQUENCE LISTINGS
(1) GENERAL INFORMATION:
(i) APPLICANT: HOLT, JEFFREY T.
JENSEN, ROY A.
PAGE, DAVID L.
OBEPMTT.T P.R, PATRICE S.
ROBINSON-BENION, CHERYL L.
THOMPSON, MARILYN E.
(ii) TITLE OF INVENTION: METHOD FOR DETECTION AND
TREATMENTS OF BREAST CANCER
(iii) NUMBER OF SEQUENCES: 49
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: I.C. WADDEY, JR.
(B) STREET: 27TH FLOOR, L & C TOWER, 401 CHURCH
(C) CITY: NASHVILLE
(D) STATE: TENNESSEE
(E) COUNTRY: USA
(F) ZIP: 37219
(v) CO~U l~K READABLE FORM:
(A) MEDIUM TYPE: Diskette, 3.50 inch, 800 kB storage
(13) CO~IJ l~K: IBM PC/XT/AT col"~alible
(C) OPERATING SYSTEM: MS-DOS (version 5.0)
(D) SOFTWARE: WordPerfect 5. l/WordPerfect Editor
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(13) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NIJMBER: U.S. 08/182,961
(B) FILING DATE: 14 JAN 1994
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(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: I.C. WADDEY, JR.
(B) REGISTRATION NUMBER: 25,180
- (C) REFERENCE/DOCKET NUMBER: 0216-9409
(ix) TELECOMMUNICATION INFORMATION (O):
(A) TELEPHONE: (615) 242-2400
(B) TELEFAX: (615) 242-2221
(C) TELEX:
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 264
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPO~l~'l'lCAL: no
(iv) ANTI-SENSE: no
(v) ORIGINAL SOURCE
(A) ORGANISM: Homo sapiens sapiens
(C) INDIVIDUAL/ISOLATE: sample of non-comedo DCIS
(D) DEVELOPMENTAL STAGE: adult
(F) TISSUE TYPE: female breast
(G) CELL TYPE: ductal carcinoma in situ
(H) CELL LINE: not derived from a cell line
ORGANELLE: no
(vii) rMMF,nIATE SOURCE:
(A) LIBRARY: cDNA library derived from human
(B) CLONE: obtained from idPntifi~ti~ n of diLrele
gene ~;A~lession
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56
(viii) POSmON IN GENOME:
(A) CHROMOSOME/SEGMENT: unknown
(B) MAP POSmON: unknown
(C) UNITS: unknown
(ix) FEATURE:
(A) NAME/KEY: DCIS-l
(B) LOCATION: (~enR~nk ~ ion no. L2736
(C) IDENTIFICATION METHOD: microscopically-directed
,~mplin~ and dirrert;"~ial display
(D) Ol~K INFORMATION: gene encoding M2 subunit of
~" " ,~ " "honucleotide redllct~ ~e
(x) PUBLICATION INFORMATION: unpublished
(K) RELEVANT RESIDUES IN SEQ ID NO: 1
(xi) SEQUENCE DESCRIPIION: SEQ ID NO:l:
TTGGGAATTG GGTACGCGGG CCCCCCACTG TGCCGAATTC CTGCATGCGG GGGATCCACT 60
AGTTCAGAGC A''"rrr~rr~r CCGTAGGACT CCAGCTTTTG I IL~.I Il,CL.I TTAGTGAGGG 120
TTMTTTTCG AG~.I lI~GCbT MTCATGGTC ATAGCTGTTT CGTGTGTGAA ATTGTTATCC 180
GCTCACAATT Cr'\''A''~ TA''r~''"rrG MGCATAAAA GTGTAAAGCC T6G~ lu~.~.l 240
MTGAGTGAG CTAACTCACA TTM 264
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 73
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOl~llCAL: no
(iv) ANTI-SENSE: no
(v) ORIGINAL SOURCE
ORGANISM: Homo sapiens sapiens
(C) INDIVIDUAL/ISOLATE: sample of non-comedo DCIS
(D) DEVELOPMENTAL STAGE: adult
(F) TISSUE TYPE: female breast
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(G) CELL TYPE: ductal carcinoma in situ
(H) CELL LINE: not derived from a cell line
(I) ORGANELLE: no
(vii) TMMFnIATE SOURCE:
(A) LIBRARY: cDNA library derived from human
(B) CLONE: obtained from irlP.ntifit~.~tion of dirrelcnlial gene
~ression
(viii) POSmON IN GENOME:
(A) CHROMOSOME/SEGMENT: unknown
(B) MAP POSlIION: unknown
(C) UNITS: unknown
(ix) FEATURE:
(A) NAME/KEY: DCIS-2
(B) LOCATION: GenR~nk ~cce~ n no. L27637
(C) IDENTIFICATION METHOD: microscopically-directed
sampling and dirre;lenlial display
(x) PUBLICATION INFORMATION: unpukli.~hed
(K) RELEVANT RESIDUES IN SEQ ID NO: 2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
TAGCCCGGTT ATCGAAATAG cr.~ r,rr TCTTCACTAT CAGCAGTACG CCGCCCAGTT 60
GTACGGACAC GGA 73
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 46
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOl H ~:llCAL: no
(iv) ANTI-SENSE: no
(v) ORIGINAL SOURCE
(A) ORGANISM: Homo sapiens sapiens
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58
(C) INDIVIDUAL/ISOLATE: sample of non-comedo DCIS
(D) DEVELOPMENTAL STAGE: adult
(F) TISSUE TYPE: female breast
(G) CELL TYPE: ductal carcinoma in situ
(H) CELL LINE: not derived from a cell line
(I) ORGANELLE: no
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: cDNA library derived from human
(13) CLONE: obtained from identification of differential gene
ression
(viii) POSIIION IN GENOME:
(A) CHROMOSOME/SEGMENT: unknown
(B) MAP POSITION: unknown .
(C) UNITS: unknown
(ix) FEATURE:
(A) NAME/KEY: DCIS-3
(B) LOCATION: L27638
(C) IDENTIFICATION METHOD: microscopically-directed
~mr~lin~ and dirr~lel~ial display
(x) PUBLICATION INFORMATION: unpublished
(K) RELEVANT RESIDUES IN SEQ ID NO: 3
(xi) SEQUENCE DESCRIPIION: SEQ ID NO: 3:
TGCCCLATGT GTGTCGTACA A~1~6CLLIG TGGCTGATTT CGAT M 46
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 72
(13) TYPE: nucleic acid "
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOl~'l'lCAL: no
(iv) ANTI-SENSE: no
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59
(v) ORIGINAL SOURCE
(A) ORGANISM: Homo sapiens sapiens
(C) INDIVIDUAL/ISOLATE: sample of non-comedo DCIS
(D) DEVELOPMENTAL STAGE: adult
(F) TISSUE TYPE: female breast
(G) CELL TYPE: ductal carcinoma in situ
(H) CELL LINE: not derived from a cell line
(I) ORGANELLE: no
(vii) TMMFnIATE SOURCE:
(A) LIBRARY: cDNA library derived from human
(B) CLONE: obtained from idçntifi~tion of dirre~,ltial gene
t;~yl~sion
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT: unknown
(B) MAP POSlTION: unknown
(C) UNITS: unknown
(ix) FEATURE:
(A) NAME/KEY: DCIS-4
(B) LOCATION: L27640
(C) IDENTIFICATION METHOD: microscopically-directed
~mplin~ and dirrer~ntial display
(x) PUBLICATION INFORMATION: unpublished
(K) RELEVANT RESIDUES IN SEQ ID NO: 4
(xi) SEQUENCE DESCRIPIION: SEQ ID NO: 4:
TAGCCCATGA b I I ~b I b ~ L~ GTACAACTGG GGC6~1 b I bG CTGATTTCGA TA' ~~ 60
ATCAGCCCGA CG 72
, (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 84
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: no
(iv) ANTI-SENSE: no
(v) ORIGINAL SOURCE
(A) ORGANISM: Homo sapiens sapiens
(C) INDIVIDUAL/ISOLATE: sample of non-comedo DCIS
(D) DEVELOPMENTAL STAGE: adult
(F) TISSUE TYPE: female breast
(G) CELL TYPE: ductal carcinoma in situ
(H) CELL LINE: not derived from a cell line
(I) ORGANELLE: no
(vii) rMMRnIATE SOURCE:
(A) LIBRARY: cDNA library derived from human
(B) CLONE: obtained from identific~tion of differential gene
expression
(viii) POSmON IN GENO~E:
(A) CHROMOSOME/SEGMENT: unknown
(B) MAP POSITION: unknown
(C) UNITS: unknown
(ix) FEATURE:
(A) NAME/KEY: DCIS-5
(B) LOCATION: L27641
(C) IDENTIFICATION METHOD: microscopically-directed
sampling and dirr~el,lial display
(x) PUBLICATION INFORMATION: unpublished
(K) RELEVANT RESIDUES IN SEQ ID NO: 5
(xi) SEQUENCE DESCRIPIION: SEQ ID NO: 5:
TAGCCCGGTT ATCGAAATCA rrr~r~GC~r CTAACTTCTG CAGAAGCCTT TGACCATCAC 60
CAGTTGTACG G~r~rlA~T CATC 84
(2) INFORMATION FOR SEQ ID NO:6:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 99
(13) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOl~l~TICAL: no
(iv) ANTI-SENSE: no
(v) ORIGINAL SOURCE
(A) ORGANISM: Homo sapiens sapiens
(C) INDIVIDUAL/ISOLATE: sample of non-comedo DCIS
(D) DEVELOPMENTAL STAGE: adult
(F) TISSUE TYPE: female breast
(G) CELL TYPE: ductal carcinoma in situ
(H) CELL LINE: not derived from a cell line
(I) ORGANELLE: no
(vii) TMM~.nIATE SOURCE:
(A) LIBRARY: cDNA library derived from human
(B) CLONE: obtained from i(lentifi~tion of differential gene
t;~.ession
(viii) POSlIION IN GENOME:
(A) CHROMOSOME/SEGMENT: unknown
(B) MAP POSITION: unknown
(C) UNITS: unknown
(ix) FEATURE:
(A) NAME/KEY: DCIS-6
(B) LOCATION: L27642
(C) IDENTIFICATION METHOD: microscopically-directed
~mpling and dirr~lell~ial display
(x) PUBLICATION INFORMATION: unpublished
(K) RELEVANT RESIDUES IN SEQ ID NO: 6
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
CCG AAATTCCTGG CM~ T GLI~GC~l~l GGAATTGTCG CG~CCC~I~G 60
CCGC66 C~llllll~l CTACATTCGT CGTAGCTCG 99
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 88
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: no
(iv) ANTI-SENSE: no
(v) ORIGINAL SOURCE
(A) ORGANISM: Homo sapiens sapiens
(C) INDIVIDUAL/ISOLATE: sample of non-comedo DCIS
(D) DEVELOPMENTAL STAGE: adult
(F) TISSUE TYPE: female breast
(G) CELL TYPE: ductal carcinoma in situ
(H) CELL LINE: not derived from a cell line
(I) ORGANELLE: no
(vii) ~MMFnIATE SOURCE:
(A) LIBRARY: cDNA library derived from human
(B) CLONE: obtained rom identification of dirr~lel.~ial gene
eA~lession
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT: unknown
(B) MAP POSITION: unknown
(C) UNITS: unknown
(iA) FEATURE:
(A) NAME/KEY: DCIS-7
(13) LOCATION: L27643
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(C) IDENTIFICATION METHOD: microscopically-directed
.~mpling and dirrclcl-tial display
(x) PUBLICATION INFORMATION: unpublished
(K) RELEVANT RESIDUES IN SEQ ID NO: 7
(xi) SEQUENCE DESCRIPrION: SEQ ID NO: 7:
ATCAGCGCGC GACATTCGGG TA-rCr~rr~rr CCCCC~I~C6 TCGGAATTCC TCG~rCr~rr' 60
ATCCATAGGA TGTGGAGTTA GTTTTGTT 88
(2) INFORMATION FOR SEQ ID NO:8
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPIION: PCR primer
(iii) HYPOl~llCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: olig-~mlc.leQtide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
cr,rr~r,rr~ GCGCb)~lbC CAGGG 25
(2) INFORMATION FOR SEQ ID NO:9
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPTION: PCR primer
(iii) HYPOTHETICAL: yes
(iv) ANTI-SENSE: no
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(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
C~LLLLILL6 TTACCCTCCC CGCCG 25
(2) INFORMATION FOR SEQ ID NO: 10
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(13) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPrION: PCR primer
(iii) HYPOTHETICAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
GGATGGCGTC CTGTAACCCG ACGCT 25
(2) INFORMATION FOR SEQ ID NO: 11
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPIION: PCR primer
(iii) Hypcrl~llcAL: yes
(iv) ANII-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
ALI~GGLILI CLI~CG~T6G CGGGG 25
(2) INFORMATION FOR SEQ ID NO: 12
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(13) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPIION: PCR primer
(iii) HYPOTHETICAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPIION: SEQ ID NO: 12:
CTGAGAGGTA ~lf~r~CCr~A GGCTG 25
(2) INFORMATION FOR SEQ ID NO:13
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPrION: PCR primer
(iii) HYPOTHETICAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIYIION: SEQ ID NO: 13:
GL~IGGCcGC GACACGGATT ACCGC 25
(2) INFORMATION FOR SEQ ID NO: 14
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
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(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPTION: PCR primer
(iii) HYPOl~llCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligon~lcleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
TTAGCGCATG GTGGACCTGG AGACG 25
(2) INFORMATION FOR SEQ ID NO: 15
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPIION: PCR primer
(iii) HYPO~ lCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPIION: SEQ ID NO: 15:
TGTGGTTACG TCAGCGAAGG TAATA 25
(2) INFORMATION FOR SEQ ID NO: 16
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPrION: PCR primer
(iii) HYPO~ llCAL: yes
(iv) ANTI-SENSE: no
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(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPIION: SEQ ID NO: 16:
AGTCGCACGC ATGTCACGCT CCGCC 25
(2) INFORMATION FOR SEQ ID NO: 17
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPTION: PCR primer
(iii) HYPOil'~'l'lCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPIION: SEQ ID NO: 17:
TATCCM GCG GCAGGCTACG AGGCC 25
(2) INFORMATION FOR SEQ ID NO: 18
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(13) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPIION: PCR primer
(iii) HYPO'l'~'l lCAL yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
r.r.~r.rr.rr~G AC6~1LI~T ATCTA 25
(2) INFORMATION FOR SEQ ID NO: 19
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nUC1eiC aCid
(C) STRANDEDNESS: Sil,g1e
(D) TOPOLOGY: 1iI1ear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPTION: PCR Primer
(iii) HYPO~ '1CAL: YeS
(iV) ANTI-SENSE: llO
(V) FRAGMENT TYPE: O1igOI1UC1eOtide
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
C I l CL I CCCC GGACTCGGGG TTAGT 25
(2) INFORMATION FOR SEQ ID NO:20
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: IIUC1eiC aCid
(C) STRANDEDNESS: Sillg1e
(D) TOPOLOGY: 1illear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPIION: PCR Primer
(iii) HYPO'1'~ 'l'lCAL: YeS
(iV) ANTI-SENSE: IIO
(V) FRAGMENT TYPE: O1igOIIUC1eOtide
(Xi) SEQUENCE DESCRIPIION: SEQ ID NO: 20:
ATGC66GC6G ~ .666~.L I G GTCGC 25
(2) INFORMATION FOR SEQ ID NO:21
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: IlUC1eiC aCid
(C) STRANDEDNESS: Sing1e
(D) TOPOLOGY linear
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(ii) MOLECULE TYPE: DNA
~,, (A) DESCRIPIION: PCR primer
(iii) HYPOl~llCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
CGTGAAGCCT AT6CCLI~L~ TCAAC 25
(2) INFORMATION FOR SEQ ID NO:22
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPIION: PCR primer
(iii) HYPOl~llCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPIION: SEQ ID NO: 22:
G~66L~I Wl AGCCCTTCAG C6ATC 25
(2) INFORMATION FOR SEQ ID NO:23
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPIION: PCR primer
(iii) HYPOl~l~lCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
-
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(xi) SEQUENCE DESCRIPIION: SEQ ID NO: 23:
GCGACACTAG GCTCCCGGAG GAGGG 25
(2) INFORMATION FOR SEQ ID NO:24
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPIION: PCR primer
(iii) HYPOl~llCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPIION: SEQ ID NO: 24:
TGr~r~rrArrC CILL~GGCCC GGTAT 25
(2) INFORMATION FOR SEQ ID NO:25
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPIION: PCR primer
(iii) HYPOl~llCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
CCGGAACTGC GATAGCGTCC GTCCC 25
(2) INFORMATION FOR SEQ ID NO:26
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPIION: PCR primer
(iii) HYPOTHETICAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPrION: SEQ ID NO: 26:
ASrSr~rC TGTTTCCCGA GAGCC 25
(2) INFORMATION FOR SEQ ID NO:27
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPIION: PCR primer
(iii) HYPOl~llCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
M CGGGTGGA CATCCGCCTG CCGCC 25
(2) INFORMATION FOR SEQ ID NO:28
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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72
(ii) MOLECULE TYPE: DNA
(A) DESCRIYIION: PCR primer
(iii) HYPOTHETICAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
TGM''rArGA TGTCMTCGT CCCGA 25
(2) INFORMATION FOR SEQ ID NO:29
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPrION: PCR primer
(iii) HYPCrl~llCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPrION: SEQ ID NO: 29:
TCATCCCCGC CCAM"~''rr TCGCC 25
(2) INFORMATION FOR SEQ ID NO:30
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPIION: PCR primer
(iii) HYPO'l'~'l'lCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
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(xi) SEQUENCE DESCRIPIION: SEQ ID NO: 30:
~ ATAGGCTGCG GCACGCGCTG GGACT 25
(2) INFORMATION FOR SEQ ID NO:31
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPIION: PCR primer
(iii) HYPOTHETICAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPIION: SEQ ID NO: 31:
GACCAGGTGC Gr~rr~fiC4T GTACA 25
(2) INFORMATION FOR SEQ ID NO:32
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(13) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPIION: PCR primer
(iii) HYPOl~'l'lCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPIION: SEQ ID NO: 32:
AGCGTAGTCA TC6G~ 6 CGCCC 25
(2) INFORMATION FOR SEQ ID NO:33
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPIION: PCR primer
(iii) HYPOl~'llCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIP~ION: SEQ ID NO: 33:
GGCCCCTAGC CCAGGGTGAA GCCCA 25
(2) INFORMATION FOR SEQ ID NO:34
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPIION: PCR primer
(iii) HYPO~ lCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonl~cl~tide
(xi) SEQUENCE DESCRIPrION: SEQ ID NO: 34:
CCCAGTGCTA cr;r.r.rrr.rrc CMGC 25
(2) INFORMATION FOR SEQ ID NO:35
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
-
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(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPTION: PCR primer
(iii) HYPOl~llCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPIION: SEQ ID NO: 35:
LL. I I L~. I l.G6 TTACCTGCCC TCGGG 25
(2) INFORMATION FOR SEQ ID NO:36
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRI~fIlON: PCR primer
(iii) HYPO~ llCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligt~n~ Qti~le
(xi) SEQUENCE DESCRIPIION: SEQ ID NO: 36:
TCCGG~4~r Arrr4rrrrA AGGGC 25
(2) INFORMATION FOR SEQ ID NO:37
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPIION: PCR primer
(iii) HYPO~ lCAL: yes
(iv) ANTI-SENSE: no
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(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPIION: SEQ ID NO: 37:
AC6CG~16~1 CC~CG~ CTGAT 25
(2) INFORMATION FOR SEQ ID NO:38
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPIION: PCR primer
(iii) HYPC)'l'~"l'lCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
CGATGCM GG CCAGCAGCAC TCGAC 25
(2) INFORMATION FOR SEQ ID NO:39
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPIION: PCR primer
(iii) HYPOl~l~lCAL: yes
(iv) ANII-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
ccc~r~rr~ r~r~rrr ACGTG 25
(2) INFORMATION FOR SEQ ID NO:40
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
- (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPTION: PCR primer
(iii) HYPO'l~'l'lCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
AGCGGGGAGG GATCGGGGGC CM GC 25
(2) INFORMATION FOR SEQ ID NO:41
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPTION: PCR primer
(iii) HYPO'l~'l'lCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCEDESCRIPTION: SEQIDNO: 41:
CC~ TA rrr~rrr~rr TCTTA 25
(2) INFORMATION FOR SEQ ID NO:42
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: DNA
(A) DESCRIPTION: PCR primer
(iii) HYPO~ lCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
CCACCCCTGT A~IGCGGG~I GCGAG 25
(2) INFORMATION FOR SEQ ID NO:43
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPIION: PCR primer
(iii) HYPCrl~llCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonllcleQtide
(xi) SEQUENCE DESCRIPIION: SEQ ID NO: 43:
rr~Arrcr~r GCCC~CCAG GGTTC 25
(2) INFORMATION FOR SEQ ID NO:44
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIYIION: PCR primer
(iii) HYPO'l~i~'l'lCAL: yes
(iv) ANTI-SENSE: no
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(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPIION: SEQ ID NO: 44:
T~r.rrr~ Ar~rrr~ GCTCC 25
(2) INFORMATION FOR SEQ ID NO:45
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPIION: PCR primer
(iii) HYPO~l'~'l~lCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPIION: SEQ ID NO: 45:
~"~''r''GGC'\~ GGGCTAGGTG GCTTA 25
(2) INFORMATION FOR SEQ ID NO:46
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(A) DESCRIPrION: PCR primer
(iii) HYPOT~llCAL: yes
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPIION: SEQ ID NO: 46:
CCG rirr~ r~ CTGCC 25
(2) INFORMATION FOR SEQ ID NO:47:
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(i) SEQUENCE CHARACTERlSTICS:
(A) LENGTH: 5712
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOl~llCAL: no
(iv) ANTI-SENSE: no
(v) ORIGINAL SOURCE
(A) ORGANISM: Homo sapiens sapiens
(C) INDIVIDUAL/ISOLATE:
(D) DEVELOPMENTAL STAGE: adult
(F) TISSUE TYPE: female breast
(G) CELL TYPE: ductal carcinoma in situ, invasive breast cancer
and normal breast tissue
(H) CELL LINE: not derived from a cell line
(I) ORGANELLE: no
(vii) TMMFnIATE SOURCE:
(A) LIBRARY: cDNA library derived from human
(B) CLONE: obtained using published sequence
(viii) POSITION IN GENOME:
(A) CHROMOSOMEtSEGMENT: unknown
(B) MAP POSITION: unknown
(C) UNITS: unknown
(ix) FEATURE:
(A) NAMEtKEY: BRCAl
(B) LOCATION: G~onR~nk accession no. U14680
(C) IDENTIFICATION METHOD: microscopically-directed
sampling and nucle~e protection assay
(D) C)1~K INFORMATION: gene encoding BRCAl protein
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(x) PUBLICATION INFORMATION:
(A) AUI~IORS: Miki, Y., et. al.
(13) IllLE: A strong c~n~ t~ gene for the breast and ovarian
cancer susceptibility gene BRCAl.
(C) JOURNAL: Science
(D) VOLUME: 266
(E) PAGES: 66-71
(F) DATE: 1994
(K) RELEVANT RESIDUES IN SEQ ID NO: 47
(xi) SEQUENCE DESCRIPIION: SEQ ID NO:47:
L~ t~9Ctyd yaCLL~Lyy acccc~ c a__ ~yLyyy yLL-~tCaya ta~tyyy~ 60
c~ycg-~ yy~yyc~-c ~ccct - ~y~t ~~yyyi ~~3 ~ y~ ~ 119
atg gat tta tct gct ctt cgc gtt gaa gaa gta caa aat gtc att aat 167
Met Asp Leu Ser Ala Leu Arg Val Glu Glu Val Gln Asn Val lle Asn
1 5 10 15
gct atg cag aaa atc tta gag tgt ccc atc tgt ctg gag ttg atc aag 215
Ala Met Gln Lys lle Leu Glu Cys Pro lle Cys Leu Glu Leu lle Lys
gaa cct gtc tcc aca aag tgt gac cac ata ttt tgc aaa ttt tgc atg 263
Glu Pro Val Ser Thr Lys Cys Asp His lle Phe Cys Lys Phe Cys Het
ctg aaa ctt ctc aac cag aag aaa 999 cct tca cag tgt cct tta tgt 311
Leu Lys Leu Leu Asn Gln Lys Lys Gly Pro Ser Gln Cys Pro Leu Cys
aag aat gat ata acc aaa agg agc cta caa gaa agt acg aga ttt agt 359
Lys Asn Asp ILe Thr Lys Arg Ser Leu Gln Glu Ser Thr Arg Phe Ser
caa ctt gtt gaa gag cta ttg aaa atc att tgt gct ttt cag ctt gac 407
Gln Leu Val Glu Glu Leu Leu Lys lle lle Cys Ala Phe Gln Leu Asp
sca ggt ttg gag tat gca aac agc tat aat ttt gca aaa aag gaa aat 455
Thr Gly Leu Glu Tyr Ala Asn Ser Tyr Asn Phe Ala Lys Lys Glu Asn
100 105 110
aac tct cct gaa cat cta aaa gat gaa gtt tct atc atc caa agt atg 503
_ Asn Ser Pro Glu Nis Leu Lys Asp Glu Val Ser lle lle Gln Ser Het
115 120 125
ggc tac aga aac cgt gcc aaa aga ctt cta cag agt gaa ccc gaa aat 551
Gly Tyr Arg Asn Arg Ala Lys Arg Leu Leu Gln Ser Glu Pro Glu Asn
13Q 135 140
cct tcc ttg cag gaa acc agt ctc agt gtc caa ctc tct aac ctt gga 5CJ9
Pro Ser Leu Gln Glu Thr Ser Leu Ser Val Gln Leu Ser Asn Leu Gly
145 150 155 160
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cact gtg aga act ctg agg aca aag cag cgg ata caa cct caa aag acg 647
Thr Val Arg Thr Leu Arg Thr Lys Gln Arg lle Gln Pro Gln Lys Thr
165 170 175
tct gtc tac att gaa ttg gga tct gat tct tct gaa gat acc gtt aat 695
Ser Val Tyr Ile Glu Leu Gly Ser Asp Ser Ser Glu Asp Thr Val Asn
180 185 190
aag gca act tat tgc agt gtg gga gat caa gaa ttg tta caa atc acc 743
Lys Ala Thr Tyr Cys Ser Val Gly Asp Gln Glu Leu Leu Gln lle Thr
195 200 205
cct caa gga acc agg gat gaa atc agt ttg gat tct gca aaa aag gct 791
Pro Gln Gly Thr Arg Asp Glu Ile Ser Leu Asp Ser Ala Lys Lys Ala
210 215 Z20
gct tgt gaa ttt tct gag acg gat gta aca aat act gaa cat cat caa 839
Ala Cys Glu Phe Ser Glu Thr Asp Val Thr Asn Thr Glu His His Gln
225 230 235 240
ccc agt aat aat gat ttg aac acc act gag aag cgt gca gct gag agg 887
Pro Ser Asn Asn Asp Leu Asn Thr Thr Glu Lys Arg Ala Ala Glu Arg
245 250 255
cat cca gaa aag tat cag ggt agt tct gtt tca aac ttg cat gtg gag 935
His Pro Glu Lys Tyr Gln Gly Ser Ser Val Ser Asn Leu His Val Glu
260 265 270
cca tgt ggc aca aat act cat gcc agc tca tta cag cat gag aac agc 983
Pro Cys Gly Thr Asn Thr His Ala Ser Ser Leu Gln His Glu Asn Ser
275 280 285
agt tta tta ctc act aaa gac aga atg aat gta gaa aag gct gaa ttc 1031
Ser Leu Leu Leu Thr Lys Asp Arg Met Asn Val Glu Lys Ala Glu Phe
290 295 300
tgt aat aaa agc aaa cag cct ggc tta gca agg agc caa cat aac aga 1079
Cys Asn Lys Ser Lys Gln Pro Gly Leu Ala Arg Ser Gln His Asn Arg
305 310 315 320
tgg gct gga agt aag gaa aca tgt aat gat agg cgg act ccc agc aca 1127
Trp Ala Gly Ser Lys Glu Thr Cys Asn Asp Arg Arg Thr Pro Ser Thr
325 330 335
gaa aaa aag gta gat ctg aat gct gat ccc ctg tgt gag aga aaa gaa 1175
Glu Lys Lys Val Asp Leu Asn Ala Asp Pro Leu Cys Glu Arg Lys Glu
340 345 350
tgg aat aag cag aaa ctg cca tgc tca gag aat cct aga gat act gaa 1223
Trp Asn Lys Gln Lys Leu Pro Cys Ser Glu Asn Pro Arg Asp Thr Glu
355 360 365
gat gtt cct tgg ata aca cta aat agc agc att cag aaa gtt aat gag 1271
Asp Val Pro Trp lle Thr Leu Asn Ser Ser lle Gln Lys Val Asn Glu
370 375 380
tgg ttt tcc aga agt gat gaa ctg tta ggt tct gat gac tca cat gat 1319
Trp Phe Ser Arg Ser Asp Glu Leu Leu Gly Ser Asp Asp Ser His Asp
385 390 395 400
ggg gag tct gaa tca aat gcc aaa gta gct gat gta ttg gac gtt cta 1367
Gly Glu Ser Glu Ser Asn Ala Lys Val Ala Asp Val Leu Asp Val Leu
405 410 415
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aat gag gta gat gaa tat tct ggt tct tca gag aaa ata gac tta ctg 1415
Asn Glu Val Asp Glu Tyr Ser Gly Ser Ser Glu Lys Ile Asp Leu Leu
420 425 430
gcc agt gat cct cat gag gct tta ata tgt aaa agt gaa aga gtt cac . 1463
Ala Ser Asp Pro His Glu Ala Leu Ile Cys Lys Ser Asp Arg VaL His
435 440 445
tcc aaa tca gta gag agt aat att gaa gac aaa ata ttt 999 aaa acc 1511
Ser Lys Ser Val Glu Ser Asp lle Glu Asp Lys Ile Phe Gly Lys Thr
450 455 460
tat cgg aag aag gca agc ctc ccc aac tta agc cat gta act gaa aat 1559
Tyr Arg Lys Lys Ala Ser Leu Pro Asn Leu Ser His Val Thr Glu Asn
465 470 475 4O0
cta att ata gga gca ttt gtt act gag cca cag ata ata caa gag cgt 1607
Leu Ile Ile Gly Ala Phe Val Ser Glu Pro Gln Ile Ile Gln Glu Arg
485 490 495
ccc ctc aca aat aaa tta aag cgt aaa agg aga cct aca tca ggc ctt 1655
Pro Leu Thr Asn Lys Leu Lys Aeg Lys Arg Arg Pro Thr Ser Gly Leu
500 505 510
cat cct gag gat ttt atc aag aaa gca gat ttg gca gtt caa aag act 1703
His Pro Glu Asp Phe Ile Lys Lys Ala Asp Leu Ala Val Gln Lys Thr
515 520 525
cct gaa atg ata aat cag gga act aac caa acg gag cag aat ggt caa 1751
Pro Glu Met Ile Asn Gln Gly Thr Asn Gln Thr Glu Gln Asn Gly Gln
530 535 540
gtg atg aat att act aat agt ggt cat gag aat aaa aca aaa ggt gat 1799
Val Met Asn Ile Thr Asn Ser Gly His Glu Asn Lys Thr Lys Gly Asp
545 550 555
tct att cag aat gag aaa aat cct aac cca ata gaa tca ctc gaa aaa 1847
Ser Ile Gln Asn Glu Lys Asn Pro Asn Pro Ile Glu Ser Leu Glu Lys
560 565 570 575
gaa tct gct ttc aaa acg aaa gct gaa cct ata agc agc agt ata agc 1895
Glu Ser Ala Phe Lys Thr Lys Ala Glu Pro Ile Ser Ser Ser Ile Ser
580 585 590
aat atg gaa ctc gaa tta aat atc cac aat tca aaa gca cct aaa aag 1943
Asn Glu Leu Glu Leu Asn Ile Het His Asn Ser Lys Ala Pro Lys Lys
595 600 605
aat agg ctg agg agg aag tct tct acc agg cat att cat gcg ctt gaa 1991
Asn Arg Leu Arg Arg Lys Ser Ser Thr Arg His Ile His Als Leu Glu
610 615 620
cta gta gtc agt aga aat cta agc cca cct aat tgt act gaa ttg caa 2039
Leu Val Val Ser Arg Asn Leu Ser Pro Pro Asn Cys Thr Glu Leu Gln
625 630 635
att gat agt tgt tct agc agt gaa gag ata aag aaa aaa aag tac aac 2087
Ile Asp Ser Cys Ser Ser Ser Glu Glu Ile Lys Lys Lys Lys Tyr Asn
640 645 650 655
caa atg cca gtc agg cac agc aga aac cta caa ctc atg gaa ggt aaa 2135
Gln Met Pro Val Arg His Ser Arg Asn Leu Gln Leu Met Glu Gly Lys
660 665 670
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gaa cct gca act gga gcc aag aag agt aac aag cca aat gaa cag aca 2183
GLu Pro Ala Thr Gly Ala Lys Lys Ser Asn Lys Pro Asn Glu Gln Thr
675 680 685
agt aaa aga cat gac agc gat act ttc cca gag ctg aag tta aca aat 2231
Ser Lys Arg His Asp Ser Asp Thr Phe Pro Glu Leu Lys Leu Thr Asn
690 695 700
gca cct ggt tct ttt act aag tgt tca aat acc agt gaa ctt aaa gaa 2279
Ala Pro Gly Ser Phe Thr Lys Cys Ser Asn Thr Ser Glu Leu Lys Glu
705 710 715
ttt gtc aat cct agc ctt cca sga gaa gaa aaa gaa gag aaa cta gaa Z327
Phe Val Asn Pro Ser Leu Pro Arg GLu Glu Lys Glu Glu Lys Leu Glu
n 0 n5 730 735
aca gtt aaa gtg tct aat aat gct gaa gac ccc aaa gat ctc atg tta 2375
Thr Val Lys Val Ser Asn Asn Ala Glu Asp Pro Lys Asp Leu Met Leu
740 745 750
agt gga gaa agg gtt ttg caa act gaa aga tct gta gag agt agc agt 2423
Ser Gly Glu Arg Val Leu Gln Thr Glu Arg Ser Val Glu Ser Ser Ser
755 760 765
att tca ttg gta cct ggt act gat tat ggc act cag gaa agt atc tcg 2471
lle Ser Leu Val Pro Gly Thr Asp Tyr Gly Thr Gln Glu Ser lle Ser
770 m 780
tta ctg gaa gtt agc act cta 999 aag gca aaa aca gaa cca aat aaa 2519
Leu Leu Glu Val Ser Thr Leu Gly Lys Ala Lys Thr Glu Pro Asn Lys
785 790 795
tgt gtg agt cag tgt gca gca ttt gaa aac ccc aag gga cta att cat 2567
Cys Val Ser Gln Cys Ala Ala Phe Glu Asn Pro Lys Gly Leu lle His
800 805 810 815
ggt tgt tcc aaa gat aat aga aat gac aca gaa ggc ttt aag tat cca 2615
Gly Cys Ser Lys Asp Asn Arg Asn Asp Thr Glu Gly Phe Lys Tyr Pro
820 825 830
ttg gga cat gaa gtt aac cac agt cgg gaa aca agc ata gaa atg gaa 2663
Leu Gly His Glu Val Asn His Ser Arg Glu Thr Ser lle Glu Met Glu
835 840 845
gaa agt gaa ctt gat gct cag tat ttg cag aat aca ttc aag gtt tca 2711
Glu Ser Glu Leu Asp Ala Gln Tyr Leu Gln Asn Thr Phe Lys Val Ser
850 855 860
aag cgc cag tca ttt gct ccg ttt tca aat cca gga aat gca gaa gag 2759
Lys Arg Gln Ser Phe Ala Pro Phe Ser Asn Pro Gly Asn Ala Glu Glu
865 870 875
gaa tgt gca aca ttc tct gcc cac tct 999 tcc tta aag aaa caa agt 2807
Glu Cys Ala Thr Phe Ser Ala His Ser Gly Ser Leu Lys Lys Gln Ser
880 885 890 895
cca aaa gtc act ttt gaa tgt gaa caa aag gaa gaa aat caa gga aag 2855
Pro Lys Val Thr Phe Glu Cys Glu Gln Lys Glu Glu Asn Gln Gly Lys
900 905 910
aat gag tct aat atc aag cct gta cag aca gtt aat atc act gca ggc 2903
Asn Glu Ser Asn lle Lys Pro Val Gln Thr Val Asn lle Thr Ala Gly
915 920 925
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ttt cct gtg gtt ggt csg aaa gat aag cca gtt gat aat gcc aaa tgt 2951
Phe Pro Val Val Gly Gln Lys Asp Lys Pro Val Asp Asn Ala Lys Cys
930 935 940
agt atc aaa gga ggc tct agg ttt tgt cta tca tct cag ttc aga ggc 2999
Ser lle Lys Gly Gly Ser Arg Phe Cys Leu Ser Ser GLn Phe Arg Gly
- 945 950 955
aac gaa act gga ctc att act cca aat aaa cat gga ctt tta caa aac 3047
Asn Glu Thr Gly Leu ILe Thr Pro Asn Lys His Gly Leu Leu Gln Asn
960 965 970 975
cca tat cgt ata cca cca ctt ttt ccc atc aag tca ttt gtt aaa act 3095
Pro Tyr Arg lle Pro Pro Leu Phe Pro lle Lys Ser Phe Val Lys Thr
980 985 990
aaa tgt aag aaa aat ctg cta gag gaa aac ttt gag gaa cat tca atg 3143
Lys Cys Lys Lys Asn Leu Leu Glu Glu Asn Phe Glu Glu His Ser Met
995 1000 1005
tca cct gaa aga gaa atg gga aat gag aac att cca agt aca gtg agc 3191
Ser Pro Glu Arg Glu Met Gly Asn Glu Asn lle Pro Ser Thr Val Ser
1010 1015 1020
aca att agc cgt aat aac att aga gaa aat gtt ttt aaa gaa gcc agc 3239
Thr lle Ser Arg Asn Asn lle Arg Glu Asn Val Phe Lys Glu Ala Ser
1025 1030 1035
tca agc aat att aat gaa gta ggt tcc agt act aat gaa gtg ggc tcc 3287
Ser Ser Asn lle Asn Glu Val Gly Ser Ser Thr Asn Glu Val Gly Ser
1040 1045 1050 1055
agt att aat gaa ata ggt tcc agt gat gaa aac att caa gca gaa cta 3335
Ser lle Asn Glu lle Gly Ser Ser Asp Glu Asn lle Gln Ala Glu Leu
1060 1065 1070
ggt aga aac aga 999 cca aaa ttg aat gct atg ctt aga tta 999 gtt 3383
Gly Arg Asn Arg Gly Pro Lys Leu Asn Ala Met Leu Arg Leu Gly Val
1075 1080 1085
ttg caa cct gag gtc tat aaa caa agt ctt cct gga agt aat tgt aa!~ 3431
Leu Gln Pro Glu Val Tyr Lys Gln Ser Leu Pro Gly Ser Asn Cys Lys
1090 1095 1100
cat cct gaa ata aaa aag caa gaa tat gaa gaa gta gtt cag act gtt 3479
His Pro Glu lle Lys Lys Gln Glu Tyr Glu Glu Val Val Gln Thr Val
1105 1110 1115
aat aca gat ttc tct cca tat ctg att tca gat aac tta gaa cag cct 3527
Asn Thr Asp Phe Ser Pro Tyr Leu lle Ser Asp Asn Leu Glu Gln Pro
1120 1125 1130 1135
atg gga agt agt cat gca tct cag gtt tgt tct gag aca cct gat gac 3575
Met Gly Ser Ser His Ala Ser Gln Val Cys Ser Glu Thr Pro Asp Asp
1140 1145 1150
ctg tta gat gat ggt gaa ata aag gaa gat act jagt ttt gct gaa aat 3623
Leu Leu Asp Asp Gly Glu lle Lys Glu Asp Thr Ser Phe Ala Glu Asn
1155 1160 1165
gac att aag gaa agt tct gct gtt ttt agc aaa agc gtc cag aaa gga 3671
Asp lle Lys Glu Ser Ser Ala Val Phe Ser Lys Ser Val Gln Lys Gly
1170 1175 1180
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gag ctt agc agg agt cct agc cct ttc acc cat aca cat ttg gct cag 3719
Glu Leu Ser Arg Ser Pro Ser Pro Phe Thr His Thr His Leu Ala Gln
1185 1190 1195
ggt tac cga aga 999 gcc aag aaa tta gag tcc tca gaa gag aac tta 3767
Gly Tyr Arg Arg Gly Ala Lys Lys Leu Glu Ser Ser Glu Glu Asn Leu
1200 1205 1210 1215
tct agt gag gat gaa gag ctt ccc tgc ttc caa cac ttg tta ttt ggt 3815
Ser Ser Glu Asp Glu Glu Leu Pro Cys Phe Gln His Leu Leu Phe Gly
1220 1225 1230
aaa gta aac aat ata cct tct cag tct act agg cat agc acc gtt gct 3863
Lys Val Asn Asn lle Pro Ser Gln Ser Thr Arg His Ser Thr Val Ala
1235 1240 1245
acc gag tgt ctg tct aag aac aca gag gag aat tta tta tca ttg aag 3911
Thr Glu Cys Leu Ser Lys Asn Thr Glu Glu Asn Leu Leu Ser Leu Lys
1250 1255 1260
aat agc tta aat gac tgc agt aac cag gta ata tts gca aag gca tct 3959
Asn Ser Leu Asn Asp Cys Ser Asn Gln Val lle Leu Ala Lys Als Ser
1265 1270 1275
cag gaa cat cac ctt agt gag gaa aca aaa tgt tct gct agc ttg ttt 4007
Gln Glu His His Leu Ser Glu Glu Thr Lys Cys Ser Ala Ser Leu Phe
1280 1285 1290 1295
tct tca cag tgc agt gaa ttg gaa gac ttg act gca aat aca aac acc 4055
Ser Ser Gln Cys Ser Glu Leu Glu Asp Leu Thr Ala Asn Thr Asn Thr
1300 1305 1310
cag gat cct ttc ttg att ggt tct tcc aaa caa atj agg cat cag tct 4103
Gln Asp Pro Phe Leu lle Gly Ser Ser Lys Gln Met Arg His Gln Ser
1315 1320 1325
gaa agc cag gga gtt ggt ctg agt gac aag gaa ttg gtt tca gat gat 4151
Glu Ser Gln Gly Val Gly Leu Ser Asp Lys Glu Leu Val Ser Asp Asp
1330 1335 1340
gaa gaa aga gga acg ggc ttg gaa gaa aat aat caa gaa gag caa agc 4199
Glu Glu Arg Gly Thr Gly Leu Glu Glu Asn Asn Gln Glu Glu Gln Ser
1345 1350 1355
atg gat tca aac tta ggt gaa gca gca tct 999 tgt gag agt gaa aca 4247
Het Asp Ser Asn Leu Gly Glu Ala Ala Ser Gly Cys Glu Ser Glu Thr
1360 1365 1370 1375
agc gtc tct gaa gac tgc tca 999 cta tcc tct cag agt gac att tta 4295
Ser Val Ser Glu Asp Cys Ser Gly Leu Ser Ser Gln Ser Asp lle Leu
13O0 1385 1390
acc act cag cag agg gat acc atg caa cat aac ctg ata aag ctc cag 4343
Thr Thr Gln Gln Arg Asp Thr Met Gln His Asn Leu lle Lys Leu Gln
1395 1400 1405
cag gaa atg gct gaa cta gaa gct gtg tta gaa cag cat 999 agc cag 4391
Gln Glu Met Ala Glu Leu Glu Ala Val Leu Glu Gln His Gly Ser Gln
1410 1415 1420
cct tct aac agc tac cct tcc atc ata agt gac tct tct gcc ctt gag 4439
Pro Ser Asn Ser Tyr Pro Ser lle lle Ser Asp Ser Ser Ala Leu Glu
1425 1430 1435
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gac ctg cga aat cca gaa caa agc aca tca gaa aaa gca gta tta act 4487
Asp Leu Arg Asn Pro Glu Gln Ser Thr Ser Glu Lys Val Leu Gln Thr
1440 1445 1450 1455
tca cag aaa agt agt gaa tac cct ata agc cag aat cca gaa ggc ctt 4535
Ser Gln Lys Ser Ser Glu Tyr Pro lle Ser Gln Asn Pro Glu Gly Xaa
~ 1460 1465 1470
tct gct gac aag ttt gag gtg tct gca gat agt tct acc agt aaa aat 4583
Ser Ala Asp Lys Phe GlU Val Ser Ala Asp Ser Ser Thr Ser Lys Asn
1475 1480 1485
aaa gaa cca gga gtg gaa agg tca tcc cct tct aaa tgc cca tca tta 4631
Lys Glu Pro Gly Val Glu Arg Ser Ser Pro Ser Lys Cys Pro Ser Leu
1490 1495 1500
gat gat agg tgg tac atg cac agt tgc tct 999 agt ctt cag aat aga 4679
Asp Asp Arg Trp Tyr Met His Ser Cys Ser Gly Ser Leu Gln Asn Arg
1505 1510 1515 1520
aac tac cca tct caa gag gag ctc att aag gtt gtt gat gtg gag gag 4727
Asn Tyr Pro Pro Gln Glu Glu Leu lle Lys Val Val Asp Val Glu Glu
1525 1530 1535
caa cag ctg gaa gag tct ggg cca cac gat ttg acg gaa aca tct tac 4775
Gln Gln Leu Glu Glu Ser Gly Pro His Asp Leu Thr Glu Thr Ser Tyr
1540 1545 1550
ttg cca agg caa gat cta gag gga acc cct tac ctg gaa tct gga atc 4823
Leu Pro Arg Gln Asp Leu Glu Gly Thr Pro Tyr Leu Glu Ser Gly lle
1555 1560 1565
agc ctc ttc tct gat gac cct gaa tct gat cct tct gaa gac aga gcc 4871
Ser Leu Phe Ser Asp Asp Pro Glu Ser Asp Pro Ser Glu Asp Arg Ala
1570 1575 1580
cca gag tca gct cgt gtt ggc aac ata cca tct tca acc tct gca ttg 4919
Pro Glu Ser Ala Arg Val Gly Asn lle Pro Ser Ser Thr Ser Ala Leu
1585 1590 1595 1600
aaa gtt ccc caa ttg aaa gtt gca gaa tct gcc cag agt cca gct gct 4967
Lys Val Pro Gln Leu Lys Val Ala Glu Ser Ala Gln Ser Pro Ala Ala
1605 1610 1615
gct cat act act gat act gct 999 tat aat gca atg gaa gaa agt gtg 5015
Ala His Thr Thr Asp Thr Ala Gly Tyr Asn Ala Met Glu Glu Ser Val
1620 1625 1630
agc agg gag aag cca gaa ttg aca gct tca aca gaa agg gtc aac aaa 5063
Ser Arg Glu Lys Pro Glu Leu Thr Ala Ser Thr Glu Arg Val Asn Lys
1635 1640 1645
aga atg tcc atg gtg gtg tct ggc ctg acc cca gaa gaa ttt atg ctc 5111
Arg Met Ser Met Val Val Ser Gly Leu Thr Pro Glu Glu Phe Met Leu
1650 1655 1660
gtg tac aag ttt gcc aga aaa cac cac atc act tta act aat cta att 5159
Val Tyr Lys Phe Ala Arg Lys His His lle Thr Leu Thr Asn Leu lle
1665 1670 1675 1680
act gaa gag act act cat gtt gtt atg aaa aca gat gct gag ttt gtg 5207
Thr Glu Glu Thr Thr His Val Val Met Lys Thr Asp Ala Glu Phe Val
1685 1690 1695
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tgt gaa cgg aca ctg saa tat ttt cta gga att gcg gga gga aaa tgg 5255
Cys Glu Arg Thr Leu Lys Tyr Phe Leu Gly lle Ala Gly Gly Lys Trp
1700 1705 1710 ~r
gta gtt agc tat ttc tgg gtg acc cag tct att aaa gaa aga aaa atg 5303
Val Val Ser Tyr Phe Trp Val Thr Gln Ser lle Lys Glu Arg Lys Het
1715 1720 1725
ctg aat gag cat gat ttt gaa gtc aga gga gat gtg gtc aat gga aga 5351
Leu ~sn Glu His ~sp Phe Glu Val ~rg Gly Asp Val Val ~sn Gly ~rg
1730 1735 1740
aac cac caa ggt cca aag cga gca aga gaa tcc cag gac aga aag atc 5399
Asn His Gln Gly Pro Lys Arg Ala Arg Glu Ser Gln Asp Arg Lys lle
1745 1750 1755 1760
ttc agg 999 cta gaa atc tgt tgc tat 999 ccc ttc acc aac atg ccc 5447
Phe Arg Gly Leu Glu lle Cys Cys Tyr Gly Pro Phe Thr Asn Het Pro
1765 1770 1m
aca gat caa ctg gaa tgg atg gta cag ctg tgt ggt gct tct gtg gtg 5495
Thr Asp Gln Leu Glu Trp Met Val Gln Leu Cys Gly Ala Ser Val Val
1780 1785 1790
aag gag ctt tca tca ttc acc ctt ggc aca ggt gtc cac cca att gtg 5543
Lys Glu Leu Ser Ser Phe Thr Leu Gly Thr Gly Val His Pro lle Val
1795 1800 1805
gtt gtg cag cca gat gcc tgg aca gag gac aat ggc ttc cat gca att 5591
Val Val Gln Pro Asp Ala Trp Tht Glu Asp Asn Gly Phe His Ala lle
1810 1815 1820
999 cag atg tgt gag gca cct gtg gtg acc cga gag tgg gtg ttg gac 5639
Gly Gln Het Cys Glu Ala Pro Val Val Thr Arg Glu Trp Val Leu Asp
1825 1830 1835 1840
agt gta gca ctc tac cag tgc cag gag ctg gac acc tac ctg ata ccc 5687
Ser Val Ala Leu Tyr Gln Cys Gln Glu Leu Asp Thr Tyr Leu lle Pro
1845 1850 1855
CA9 atc ccc cac agc cac tac tgat 5712
Gln lle Pro His Ser His Tyr
1860
(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1237
~) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA regulatory sequence 4
(iii) HYPOl~llCAL: no
(iv) ANTI-SENSE: no
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(v) ORIGINAL SOURCE
(A) ORGANISM: Homo sapiens sapiens
(C) INDIVIDUAL/ISOLATE:
(D) DEVELOPMENTAL STAGE: adult
(F) TISSUE TYPE: female breast
(G) CELL TYPE: normal breast
(H) CELL LINE: not derived from a cell line
(I) ORGANELLE: no
(vii) TMMF.r~IATE SOURCE:
(A) LIBRARY: cDNA library derived from human
(B) CLONE: obtained using published sequence
(viii) POSmON IN GENOME:
(A) CHROMOSOME/SEGMENT: unknown
(B) MAP POSITION: unknown
(C) UNITS: unknown
(ix) FEATURE:
(A) NAME/KEY: BRCAl promoter
(B) LOCATION:
(C) IDENTIFICATION METHOD: restriction enzyme digest
(D) Ol~K INFORMATION: DNA sequence regulating gene
encoding BRCAl protein
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Brown et al.
(B) TITLE: Scien~ific Correspondence
(C) JOURNAL: Nature
(D) VOLUME: 372
(E) PAGES: 733
(F) DATE: 22/29 DEt~.FMRF.~ 1994
(K) RELEVANT RESIDUES IN SEQ ID NO: 48
(xi) SEQUENCE DESCRIPIION: SEQ ID NO:48:
TTCCGGGACT CTACTACCTT TA~rr~ G AGAGGGTGAA GGC~IL~lGA TCrr~ r 60
CCAGTTATCT C~AA~rCC ACAGCCTGGT CC66G~I~LA GGAAGTCTCA GCGAGCTCAC 120
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r~r~rrrrr-~rT CGCAGTTTTA ATTTATCTGT MTTCCCGCG 1,111 I Ll,l.I I rrr~~r~GLAAA 180
Cr~'\rrrrrT ACCGCTMGC AGCAGCCTCT CAGMTACGA MTCMGGTA CMTCAGAGG 240
Mrrr~rrr~ r4r~Mr~r CMGCGTCTC TCG66CI,ll.;l GGATTGGCCA CCCAGTCTGC 300
CCCCGGATGA CGTMMGGA AAr~r~~rr'\ Ar~~r~r'\A TTCTACCTGA bl lL.I~CCl,lA 360
AArrrrrrrr CL. I l; I I,I~C~. I CTACGCTTCC Ab l l GCGb-. I TATTACGTCA CAGTMTTGC 420
TGTACCMGG TCAGAATCGC CACCTGAGGC CTGMTATCA GCGTAAGATA GTGTCCMMG 480
CAGTCTTMG MGAGGTCCC ATTACCCCAC l~;l l lI,(,GL-. CTMTGGAGT CCTCCAGTTT 540
AGGTAMTM MGGATTGTT GGGAGGTGGA Crr~Mr~r TACTATTTCC MCATGCATT 600
rrrGMrr~A AGGCLI lGGC CACACTGTTC CTTGGAMCT GTAGTCTTAT Gr'\r'~rr~/\r 660
ATCCMTACC MArrrGGCA CMTTCTCAC GGAMTCCAG TGGATAGATT GGAGACCTCC 720
GCGGGCTTAT ACATGTCMC AGTMTATTG GGTTGTTATG TTCTCCTATC TTGAGAGCAG 780
AGACTAGGCC AMAAMGAT ATAGGMGAC TACGATTCCC ATCCAGCCCC ACGAGTCTCG 840
GGCMGTAGT CCTCTMGGT CAGTGGCCTG CÇGrG~~rr~ blbGGCGCCG MTTTGCCTG 900
Cr~'\Arr5GA MTCCCTCTC TGGTCACATC TGCGCACTCC TAGTTCCGCC CCTCAGCATC 960
MTGTTTGTT Al Ibl Ibl IC GGGTTCAGGT Tb~ ;lbCC CCGCCCCATC GACGCMTCT 1020
CCACCMTCA AT6GCl.lbl.l CGTTTTGAGG GACMGTGGT r'\r~rrCMT CATCTTGGCG 1080
MCACTCGGA t'/~AA-4rrrr ACTAGTTACT GTCTTTATCC GCCATGTTAG ATTCACCCCA 1140
CAGGGATAGC GGC~'''\rrrG GTAGCGGACG liILI,I IbCAT TGGC~.IL~.l G c4rrrr~rcrc 1200
C~G6rrr~rr~r~ GMGCTGGTA A~C~~r4rr TGCGGTT 1237
(2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1863
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOl~llCAL: no
(iv) ANTI-SENSE: no
(v) ORIGINAL SOURCE
(A) ORGANISM: Homo sapiens sapiens
(C) INDIVIDUAL/ISOLATE:
(D) DEVELOPMENTAL STAGE: adult
(F) TISSUE TYPE: female breast
(G) CELL TYPE: normal breast tissue
(H) CELL LINE: not derived from a cell line
(I) ORGANELLE: no
(ix) FEATURE:
(A) NAME/KEY: BRCAl protein
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2 (B) LOCATION: 1 to 1863
- (C) IDENTIFICATION METHOD: observation of mRNA and
~nri~Pn~e. inhibition of BRCAl gene
- (D) O1'~K INFORMATION: BRCAl protein has a negative
regulatory effect on growth of human m~mm~ry cells.
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Miki, Y., et. al.
(B) TlILE: A strong c~n~ te gene for the breast and ovarian
cancer susceptibility gene BRCAl.
(C) JOURNAL: Science
(D) VOLUME: 266
(E) PAGES: 66-71
(F) DATE: 1994
(K) RELEVANT RESIDUES IN SEQ ID NO: 49
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
Met Asp Leu Ser Ala Leu Arg V8l Glu Glu Val Gln Asn Val I le Asn
1 5 10 15
Ale Het Gln Lys I le Leu Glu Cys Pro I le Cys Leu Glu Leu I le Lys
Glu Pro Val Ser Thr Lys Cys Asp His lle Phe Cys Lys Phe Cys Met
Leu Lys Leu Leu Asn Gln Lys Lys Gly Pro Ser Gln Cys Pro Leu Cys
Lys Asn Asp lle Thr Lys Arg Ser Leu Gln Glu Ser Thr Arg Phe Ser
Gln Leu Val Glu Glu Leu Leu Lys Ile lle Cys Ala Phe Gln Leu Asp
Thr Gly Leu Glu Tyr Ala Asn Ser Tyr Asn Phe Ala Lys Lys Glu Asn
100 105 110
Asn Ser Pro Glu His Leu Lys Asp Glu Val Ser lle lle Gln Ser Met
115 120 125
Gly Tyr Arg Asn Arg Ala Lys Arg Leu Leu Gln Ser Glu Pro Glu Asn
130 135 140
Pro Ser Leu Gln Glu Thr Ser Leu Ser Val Gln Leu Ser Asn Leu Gly
145 150 155 160
Thr Val Arg Thr Leu Arg Thr Lys Gln Arg lle Gln Pro Gln Lys Thr
165 170 175
Ser Val Tyr lle Glu Leu Gly Ser Asp Ser ser Glu Asp Thr Val Asn
180 185 190
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Lys Ala Thr Tyr Cys Ser Val Gly Asp Gln Glu Leu Leu Gln lle Thr
195 200 205
Pro Gln Gly Thr Arg Asp Glu lle Ser Leu Asp Ser Ala Lys Lys Ala
210 215 220
Ala Cys Glu Phe Ser Glu Thr Asp Val Thr Asn Thr Glu His His Gln
225 230 235 240
Pro Ser Asn Asn Asp Leu Asn Thr Thr Glu Lys Arg Ala Ala Glu Arg
245 250 255
His Pro Glu Lys Tyr Gln Gly Ser Ser Val Ser Asn Leu His Val Glu
260 265 270
Pro Cys Gly Thr Asn Thr His Ala Ser Ser Leu Gln His Glu Asn Ser
275 280 285
Ser Leu Leu Leu Thr Lys Asp Arg Met Asn Val Glu Lys Ala Glu Phe
290 295 300
Cys Asn Lys Ser Lys Gln Pro Gly Leu Ala Arg Ser Gln His Asn Arg
305 310 315 320
Trp Ala Gly Ser Lys Glu Thr Cys Asn Asp Arg Arg Thr Pro Ser Thr
325 330 335
Glu Lys Lys Val Asp Leu Asn Ala Asp Pro Leu Cys Glu Arg Lys Glu
340 345 350
Trp Asn Lys Gln Lys Leu Pro Cys Ser Glu Asn Pro Arg Asp Thr Glu
355 360 365
Asp Val Pro Trp lle Thr Leu Asn Ser Ser lle Gln Lys Val Asn Glu
370 375 380
Trp Phe Ser Arg Ser Asp Glu Leu Leu Gly Ser Asp Asp Ser His Asp
385 390 395 400
Gly Glu Ser Glu Ser Asn Ala Lys Val Ala Asp Val Leu Asp Val Leu
405 410 415
Asn Glu Val Asp Glu Tyr Ser Gly Ser Ser Glu Lys lle Asp Leu Leu
420 425 430
Ala Ser Asp Pro His Glu Ala Leu lle Cys Lys Ser Asp Arg Val His
435 440 445
Ser Lys Ser Val Glu Ser Asp lle Glu Asp Lys lle Phe Gly Lys Thr
450 455 460
Tyr Arg Lys Lys Ala Ser Leu Pro Asn Leu Ser His Val Thr Glu Asn
465 470 475 480
Leu lle lle Gly Ala Phe Val Ser Glu Pro Gln lle lle Gln Glu Arg
485 490 495
Pro Leu Thr ~sn Lys Leu Lys Aeg Lys Arg Arg Pro Thr Ser Gly Leu
500 505 510
His Pro Glu Asp Phe lle Lys Lys Ala Asp Leu Ala Val Gln Lys Thr
515 520 525
Pro Glu Met lle Asn Gln Gly Thr Asn Gln Thr Glu Gln Asn Gly Gln
530 535 540
Val Met Asn lle Thr Asn Ser Gly His Glu Asn Lys Thr Lys Gly Asp
545 550 555
Ser lle Gln Asn Glu Lys Asn Pro Asn Pro lle Glu Ser Leu Glu Lys
560 565 570 575
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Glu ser Ala Phe Lys Thr Lys ALa Glu Pro lle Ser Ser Ser lle Ser
580 585 590
Asn Glu Leu Glu Leu Asn lle Het His Asn Ser Lys Ala Pro Lys Lys
595 600 605
Asn Arg Leu Arg Arg Lys Ser Ser Thr Arg His lle His Als Leu Glu
610 615 620
Leu Val Val Ser Arg Asn Leu Ser Pro Pro Asn Cys Thr Glu Leu Gln
625 630 635
lle Asp Ser Cys Ser Ser Ser Glu Glu lle Lys Lys Lys Lys Tyr Asn
640 645 650 655
Gln Met Pro Val Arg His Ser Arg Asn Leu Gln Leu Met Glu Gly Lys
660 665 670
Glu Pro Ala Thr Gly Ala Lys Lys Ser Asn Lys Pro Asn Glu Gln Thr
675 680 685
Ser Lys Arg His Asp Ser Asp Thr Phe Pro Glu Leu Lys Leu Thr Asn
690 695 700
Ala Pro Gly Ser Phe Thr Lys Cys Ser Asn Thr Ser Glu Leu Lys Glu
705 710 715
Phe Val Asn Pro Ser Leu Pro Arg Glu Glu Lys Glu Glu Lys Leu Glu
no ns 730 735
Thr Val Lys Vsl Ser Asn Asn Ala Glu Asp Pro Lys Asp Leu Met Leu
740 745 750
Ser Gly Glu Arg Val Leu Gln Thr Glu Arg Ser Val Glu Ser Ser Ser
755 760 765
lle Ser Leu Val Pro Gly Thr Asp Tyr Gly Thr Gln Glu Ser lle Ser
770 775 780
Leu Leu Glu Val Ser Thr Leu Gly Lys Ala Lys Thr Glu Pro Asn Lys
785 790 795
Cys Val Ser Gln Cys Ala Ala Phe Glu Asn Pro Lys Gly Leu lle His
800 805 810 815
Gly Cys Ser Lys Asp Asn Arg Asn Asp Thr Glu Gly Phe Lys Tyr Pro
820 825 830
Leu Gly His Glu Val Asn His Ser Arg Glu Thr Ser lle Glu ~et Glu
835 840 845
Glu Ser Glu Leu Asp Ala Gln Tyr Leu Gln Asn Thr Phe Lys Val Ser
850 855 860
Lys Arg Gln Ser Phe Ala Pro Phe Ser Asn Pro Gly Asn Ala Glu Glu
865 870 875
Glu Cys Ala Thr Phe Ser Ala His Ser Gly Ser Leu Lys Lys Gln Ser
880 885 890 895
Pro Lys Vsl Thr Phe Glu Cys Glu Gln Lys Glu Glu Asn Gln Gly Lys
900 905 910
Asn Glu Ser Asn lle Lys Pro Val Gln Thr Val Asn lle Thr Ala Gly
915 920 925
Phe Pro Val Val Gly Gln Lys Asp Lys Pro Val Asp Asn Ala Lys Cys
930 935 940
Ser lle Lys Gly Gly Ser Arg Phe Cys Leu Ser Ser Gln Phe Arg Gly
945 950 955
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Asn Glu Thr Gly Leu lle Thr Pro Asn Lys His Gly Leu Leu Gln Asn
960 965 970 975~ro Tyr Arg lle Pro Pro Leu Phe Pro lle Lys Ser Phe Val Lys Thr
980 985 990~ys Cys Lys Lys Asn Leu Leu Glu Glu Asn Phe Glu Glu His Ser Met
995 1000 1005
Ser Pro Glu Arg Glu Met Gly Asn Glu Asn lle Pro Ser Thr Val Ser
1010 1015 1020
Thr lle Ser Arg Asn Asn lle Arg Glu Asn Val Phe Lys Glu Ala Ser
1025 1030 1035
Ser Ser Asn lle Asn Glu Val Gly Ser Ser Thr Asn Glu Val Gly Ser
1040 1045 1050 1055~er lle Asn Glu lle Gly Ser Ser Asp Glu Asn lle Gln Ala Glu Leu
1060 1065 1070~ly Arg Asn Arg G~y Pro Lys Leu Asn Ala Met Leu Arg Leu Gly Val
1075 1080 10O5
Leu Gln Pro Glu Val Tyr Lys Gln Ser Leu Pro Gly Ser Asn Cys Lys
1090 1095 1100
His Pro Glu lle Lys Lys Gln Glu Tyr Glu Glu Val Val Gln Thr Val
1105 1110 1115
Asn Thr Asp Phe Ser Pro Tyr Leu lle Ser Asp Asn Leu Glu Gln Pro
1120 1125 1130 1135~et Gly Ser Ser His Ala Ser Gln Val Cys Ser Glu Thr Pro Asp Asp
1140 1145 1150~eu Leu Asp Asp Gly Glu lle Lys Glu Asp Thr Ser Phe Ala Glu Asn
1155 1160 1165
Asp lle Lys Glu Ser Ser Ala Val Phe Ser Lys Ser Val Gln Lys Gly
1170 1175 1180
Glu Leu Ser Arg Ser Pro Ser Pro Phe Thr His Thr His Leu Ala Gln
1185 1190 1195
Gly Tyr Arg Arg Gly Ala Lys Lys Leu Glu Ser Ser Glu Glu Asn Leu
1200 1Z05 1210 1215~er Ser Glu Asp Glu Glu Leu Pro Cys Phe Gln His Leu Leu Phe Gly
1220 1225 1230~ys Val Asn Asn lle Pro Ser Gln Ser Thr Arg His Ser Thr Val Ala
1235 1240 1245
Thr Glu Cys Leu Ser Lys Asn Thr Glu Glu Asn Leu Leu Ser Leu Lys
1250 1255 1260
Asn Ser Leu Asn Asp Cys Ser Asn Gln Val lle Leu Ala Lys Als Ser
1265 1270 1275
Gln Glu His His Leu Ser Glu Glu Thr Lys Cys Ser Ala Ser Leu Phe
1280 1285 1290 1295~er Ser Gln Cys Ser Glu Leu Glu Asp Leu Thr Ala Asn Thr Asn Thr
1300 1305 1310~ln Asp Pro Phe Leu lle Gly Ser Ser Lys Gln Met Arg His Gln Ser
1315 1320 1325~lu Ser Gln Gly Val Gly Leu Ser Asp Lys Glu Leu Val Ser Asp Asp
1330 1335 1340
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Glu Glu Arg Gly Thr Gly Leu Glu Glu Asn Asn Gln Glu Glu Gln ser
1345 1350 1355
Met Asp Ser Asn Leu Gly Glu Ala Ala Ser Gly Cys Glu Ser Glu Thr
1360 1365 1370 1375~er Val Ser Glu Asp Cys Ser Gly Leu Ser Ser Gln Ser Asp lle Leu
1380 1385 1390~hr Thr Gln Gln Arg Asp Thr Met Gln His Asn Leu lle Lys Leu Gln
1395 1400 1405
Gln Glu Met Ala Glu Leu Glu Ala Val Leu Glu Gln His Gly Ser Gln
1410 1415 1420
Pro Ser Asn Ser Tyr Pro Ser lle lle Ser Asp Ser Ser Ala Leu Glu
1425 1430 1435
Asp Leu Arg Asn Pro Glu Gln Ser Thr Ser Glu Lys Val Leu Gln Thr
1440 1445 1450 1455~er Gln Lys Ser Ser Glu Tyr Pro lle Ser Gln Asn Pro Glu Gly Xaa
1460 1465 1470
Ser Ala Asp Lys Phe Glu Val Ser Ala Asp Ser Ser Thr Ser Lys Asn
1475 1480 1485
Lys Glu Pro Gly Val Glu Arg Ser Ser Pro Ser Lys Cys Pro Ser Leu
1490 1495 1500
Asp Asp Arg Trp Tyr Met His Ser Cys Ser Gly Ser Leu Gln Asn Arg
1505 1510 1515 1520~sn Tyr Pro Pro Gln Glu Glu Leu lle Lys Val Val Asp Val Glu Glu
1525 1530 1535~ln Gln Leu Glu Glu Ser Gly Pro His Asp Leu Thr Glu Thr Ser Tyr
1540 1545 1550
Leu Pro Arg Gln Asp Leu Glu Gly Thr Pro Tyr Leu Glu Ser Gly lle
1555 1560 1565
Ser Leu Phe Ser Asp Asp Pro Glu Ser Asp Pro Ser Glu Asp Arg Ala
1570 1575 1580
Pro Glu Ser Ala Arg Val Gly Asn lle Pro Ser Ser Thr Ser Ala Leu
1585 1590 1595 1600~ys Val Pro Gln Leu Lys Val Ala Glu Ser Ala Gln Ser Pro Ala Ala
1605 1610 1615~la His Thr Thr Asp Thr Ala Gly Tyr Asn Ala Met Glu Glu Ser Val
1620 1625 1630
Ser Arg Glu Lys Pro Glu Leu Thr Ala Ser Thr Glu Arg Val Asn Lys
1635 1640 1645
Arg Met Ser Met Val Val Ser Gly Leu Thr Pro Glu Glu Phe Met Leu
1650 1655 1660
Val Tyr Lys Phe Ala Arg Lys His His lle Thr Leu Thr Asn Leu lle
1665 1670 1675 1680~hr Glu Glu Thr Thr His Val Val Met Lys Thr Asp Ala Glu Phe Val
1685 1690 1695~ys Glu Arg Thr Leu Lys Tyr Phe Leu Gly lle Ala Gly Gly Lys Trp
1700 1705 1710~al Val Ser Tyr Phe Trp Val Thr Gln Ser lle Lys Glu Arg Lys Met
1715 1720 1725
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Leu Asn Glu His Asp Phe Glu VaL Arg Gly Asp V8l Val Asn Gly Arg
1730 1735 1740
Asn His Gln Gly Pro Lys Arg Ala Arg Glu Ser Gln Asp Arg Lys lle r
1745 1750 1755 1760
Phe Arg Gly Leu Glu lle Cys Cys Tyr Gly Pro Phe Thr Asn Met Pro
1765 1770 1775
Thr Asp Gln Leu Glu Trp ~4et Val Gln Leu Cys Gly Ala Ser Val Val
178O 1785 1790
Lys Glu Leu Ser Ser Phe Thr Leu Gly Thr Gly Val His Pro lle Val
1795 1800 1805
Val V8l Gln Pro Asp Ala Trp Tht Glu Asp Asn Gly Phe His Ala lle
1810 1815 1820
Gly Gln ~et Cys Glu Ala Pro Val Val Thr Arg Glu Trp Val Leu Asp
1825 1830 1835 1840
Ser Val Ala Leu Tyr Gln Cys Gln Glu Leu Asp Thr Tyr Leu lle Pro
1845 1850 1855
Gln lle Pro His Ser His Tyr
1860
