U.S. patent application number 11/254841 was filed with the patent office on 2007-06-21 for mum-1 protein expressed by multiple myeloma-related gene.
This patent application is currently assigned to The Trustees of Columbia University In The City of New York. Invention is credited to Riccardo Dalla-Favera.
Application Number | 20070141579 11/254841 |
Document ID | / |
Family ID | 24625029 |
Filed Date | 2007-06-21 |
United States Patent
Application |
20070141579 |
Kind Code |
A1 |
Dalla-Favera; Riccardo |
June 21, 2007 |
MUM-1 protein expressed by multiple myeloma-related gene
Abstract
This invention provides a method of determining a chromosomal
breakpoint in a subject suffering from multiple myeloma which
comprises steps of: (a) obtaining a DNA sample from the subject
suffering from multiple myeloma; (b) determining whether there is J
and C disjunction in the immunoglobulin heavy chain gene in the
obtained DNA sample; (c) obtaining a genomic library having clones
which contain genomic DNA fragments from the DNA sample which shows
positive J and C disjunction; (d) selecting and isolating clones of
the obtained library which show positive hybridization with a probe
which is capable of specifically hybridizing with the C but not the
J region of the immunoglobulin heavy chain gene; (e) preparing
fluorescent probes from the genomic DNA fragments of the isolated
clones from step (d); (f) hybridizing said fluorescent probes with
metaphase chromosomes; and (g) determining the identity of the
chromosomes which are capable of hybridizing to said fluorescent
probes, wherein the identification of a chromosome other than
chromosome 14 would indicate that the chromosomal breakpoint is
between chromosome 14 and the identified chromosome, thereby
determining a chromosomal breakpoint in a subject suffering from
multiple myeloma. This invention also provides the identified gene
altered by a chromosomal breakpoint and various uses thereof.
Inventors: |
Dalla-Favera; Riccardo; (New
York, NY) |
Correspondence
Address: |
COOPER & DUNHAM, LLP
1185 AVENUE OF THE AMERICAS
NEW YORK
NY
10036
US
|
Assignee: |
The Trustees of Columbia University
In The City of New York
|
Family ID: |
24625029 |
Appl. No.: |
11/254841 |
Filed: |
October 20, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09585023 |
Jun 1, 2000 |
6958386 |
|
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11254841 |
Oct 20, 2005 |
|
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08654482 |
May 28, 1996 |
6245562 |
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09585023 |
Jun 1, 2000 |
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Current U.S.
Class: |
435/6.14 |
Current CPC
Class: |
C07K 16/32 20130101;
C07K 14/82 20130101; A61K 38/00 20130101; A61P 35/00 20180101; C07K
14/4702 20130101; A61P 35/02 20180101 |
Class at
Publication: |
435/006 |
International
Class: |
C40B 30/06 20060101
C40B030/06; C40B 40/08 20060101 C40B040/08; C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
[0001] The invention disclosed herein was made with Government
support under NIH Grant No. CA 44025. Accordingly, the U.S.
[0002] Government has certain rights in this invention.
Claims
1-89. (canceled)
90. An antibody directed to a purified MUM-1 protein.
91. An antibody capable of specifically recognizing MUM-1 protein,
wherein the MUM-1 protein has an amino acid sequence as set forth
in SEQ ID NO:14.
92. The antibody of claim 91, wherein the MUM-1 protein is a human
MUM-1 protein.
93-97. (canceled)
98. A monoclonal antibody of claim 90.
99-101. (canceled)
102. A monoclonal antibody of claim 91.
103. A monoclonal antibody of claim 92.
Description
BACKGROUND OF THE INVENTION
[0003] Throughout this application, various references are referred
to within parentheses. Disclosures of these publications in their
entireties are hereby incorporated by reference into this
application to more fully describe the state of the art to which
this invention pertains. Full bibliographic citation for these
references may be found at the end of this application, preceding
the claims.
[0004] Multiple myeloma (MM) is an incurable B cell tumor affecting
B cell end-stage differentiation. Clinically, the course of MM is
similar to end-stage plasma cell leukemia (PCL), i.e., there is an
uncontrollable proliferation of myeloma cells accompanied by
numerous complications, including hyperviscosity syndromes,
hypercalcemia, infections, multiple bone fractures, and organ
failure.
[0005] Non-random chromosomal translocation is known to play a
crucial role in the tumorigenesis of hematologic malignancies (1).
In B-cell lymphomas, many important proto-oncogenes deregulated by
juxtaposition to immunoglobulin (Ig) gene locus have been
identified. Each proto-oncogene is associated with a specific
subtype of lymphoma, such as c-MYC in Burkitt's lymphoma, Cyclin DI
IBCLI in mantle cell lymphoma, BCL-2 in follicular lymphoma and
BCL-6 in diffuse large cell lymphoma (2-8). In contrast, little is
known about molecular alterations of human MM/PCL, due to the
difficulty in cytogenetic analysis. However, previous cytogenetic
reports have shown a 14q+chromosome, suggesting the existence of a
chromosomal translocation involving the Ig heavy chain (IgH) locus,
is observed in 20.about.30% of the MM/PCL cases and it is the most
frequent consistent abnormality (9-12). Even in such cases, most
cytogenetic data have failed to identify donor chromosomes other
than llql3, 8q24, and 18q21, where proto-oncogenes Cyclin
DIIBCL-IIPRADI, c-MYC and BCL-2 are located, respectively. Among
them, the llql3 locus has been demonstrated to be involved in
nearly 5.about.10% of the cases and also in 62% of the established
cell lines (13). The t(11;14) (ql3;q32) translocation is also
accompanied by a corresponding overexpression of the Cyclin Dl
gene, which raises a strong possibility of the involvement of this
gene, although the breakpoints at llql3 do not cluster like those
of the lymphoma cases (14-16). Recent advances in fluorescence in
situ hybridization (FISH) have made it possible to clarify both the
frequency of the 14q+chromosomes and the partner chromosomes of the
IgH loci. One such report revealed an intriguing result, i.e., that
numerous chromosomal loci are able to translocate to IgH locus,
including 6p21, lq21, 3pl1, 7ql1, 1lq23 (17). This has prompted a
search for the proto-oncogenes deregulated by the regulatory
elements of the IgH gene for a further understanding of the
molecular mechanisms of MM/PCL. In the present study, one candidate
proto-oncogene, MUM1 (multiple myeloma oncogene 1), was found
juxtaposed to the IgH gene as a result of t(6;14)(p25; q32)
translocation in human myeloma cell line, SKMM-1. Over expression
of the MUM1 mRNA was observed in this cell line. A second gene,
called MUM-2 was found translocated in proximity to the IgH gene on
chromosome 14q32 in human myeloma cell line, U-266.
[0006] The method of analysis of 14q+chromosomal translocations and
identification of the genes altered in multiple myeloma of this
invention are useful since 1) no method is currently available to
determine the chromosomal sequences involved in 14q+translocations,
the most important cytogenetic lesions associated with MM
pathogenesis; 2) no specific gene lesion is currently known for MM;
3) no diagnostic method based on gene/DNA lesion is currently
available for MM and 4) there are no therapeutic approaches aimed
at counteracting the action of abnormal gene products in MM.
SUMMARY OF THE INVENTION
[0007] This invention provides a method of determining a
chromosomal breakpoint in a subject suffering from multiple myeloma
which comprises steps of: (a) obtaining a DNA sample from the
subject suffering from multiple myeloma; (b) determining whether
there is J and C disjunction in the immunoglobulin heavy chain gene
in the obtained DNA sample; (c) obtaining a genomic library having
clones which contain genomic DNA fragments from the DNA sample
which shows positive J and C disjunction; (d) selecting and
isolating clones of the obtained library which show positive
hybridization with a probe which is capable of specifically
hybridizing with the C but not the J region of the immunoglobulin
heavy chain gene; (e) preparing fluorescent probes from the genomic
DNA fragments of the isolated clones from step (d); (f) hybridizing
said fluorescent probes with metaphase chromosomes; and (g)
determining the identity of the chromosomes which are capable of
hybridizing to said fluorescent probes, wherein the identification
of a chromosome other than chromosome 14 would indicate that the
chromosomal breakpoint is between chromosome 14 and the identified
chromosome, thereby determining a chromosomal breakpoint in a
subject suffering from multiple myeloma.
[0008] This invention provides a method to identify a gene other
than the immunoglobulin gene which is located in chromosome 14,
altered by a chromosomal breakpoint detected in a subject suffering
from multiple myeloma which comprises steps of: a) selecting a
probe having a sequence of a chromosome other than chromosome 14,
identified at the chromosomal breakpoint detected in a subject
suffering from multiple myeloma, wherein said probe is capable of
hybridizing to the unique sequence of the gene other than the
immunoglobulin gene altered by a chromosomal breakpoint detected in
a subject suffering from multiple myeloma; b) contacting said probe
with mRNA isolated from a cell under conditions permitting
formation of a complex between said probe and the mRNA; c)
isolating the complex resulting from step (b); d) determining the
sequence of the mRNA in the isolated complex, thereby determining
the identity of the gene.
[0009] This invention provides a gene designated MUM-1. This
invention provides a gene designated MUM-2. This invention provides
an isolated nucleic acid molecule encoding a MUM protein. This
invention provides a DNA encoding a MUM protein. This invention
provides a cDNA encoding a MUM protein. This invention provides a
genomic DNA molecule encoding a MUM protein. This invention
provides a RNA molecule encoding a MUM protein. This invention
provides an isolated nucleic acid molecule encoding a human MUM-1
protein. This invention provides an isolated nucleic acid molecule
encoding a human MUM-2 protein. This invention provides an isolated
nucleic acid molecule encoding a MUM protein operatively linked to
a promoter of RNA transcription. This invention provides a vector
comprising the an isolated cDNA encoding a MUM protein. This
invention provides a vector which comprises an isolated cDNA
encoding a MUM protein. This invention provides a vector which
comprises an isolated cDNA encoding a MUM protein, wherein the
vector is a plasmid. This invention provides a host cell for the
vector which comprises an isolated cDNA encoding a MUM protein.
[0010] This invention provides a nucleic acid probe comprising a
nucleic acid molecule of at least 15 nucleotides capable of
specifically hybridizing with a unique sequence included within the
sequence of a nucleic acid molecule encoding a MUM protein. This
invention provides a nucleic acid probe comprising a nucleic acid
molecule of at least 15 nucleotides which is complementary to a
sequence of the isolated nucleic acid molecule encoding a MUM
protein.
[0011] This invention provides a nucleic acid probe comprising a
nucleic acid molecule of at least 15 nucleotides which is
complementary to a sequence of the isolated nucleic acid molecule
encoding a MUM protein which is linked to a nucleic acid sequence
complementary to a sequence of a nucleic acid molecule of human
chromosome 14.
[0012] This invention provides a nucleic acid probe comprising a
the sequence of a nucleic acid molecule encoding a MUM-1 protein
which is linked at a specific break point to a specified nucleic
acid sequence of human chromosome 14.
[0013] This invention provides a nucleic acid probe comprising a
the sequence of a nucleic acid molecule encoding a MUM-2 protein
which is linked at a specific break point to a specified nucleic
acid sequence of human chromosome 14.
[0014] This invention provides a method for detecting a
predisposition to multiple myeloma associated with the expression
of a human MUM-1 protein in a sample from a subject which comprises
detecting in a sample from the subject a rearrangement of nucleic
acid encoding MUM-1 protein. This invention provides a method for
detecting a predisposition to multiple myeloma associated with the
expression of a human MUM-2 protein in a sample from a subject
which comprises detecting in a sample from the subject a
rearrangement of nucleic acid encoding MUM-2 protein.
[0015] This invention provides an antisense oligonucleotide having
a sequence capable of specifically hybridizing to an mRNA molecule
encoding a human MUM-1 protein so as to prevent overexpression of
the mRNA molecule. This invention provides an antisense
oligonucleotide having a sequence capable of specifically
hybridizing to an mRNA molecule encoding a human MUM-2 protein so
as to prevent overexpression of the mRNA molecule.
[0016] This invention provides an antisense oligonucleotide having
a sequence capable of specifically hybridizing to an isolated cDNA
molecule encoding a MUM protein. This invention provides an
antisense oligonucleotide having a sequence capable of specifically
hybridizing to the isolated genomic DNA molecule encoding a MUM
protein. This invention provides an antisense oligonucleotide
having a sequence capable of specifically hybridizing to an
isolated RNA molecule encoding a MUM protein.
[0017] This invention provides a purified MUM protein. This
invention provides a purified MUM-1 protein. This invention
provides an antibody directed to a purified MUM-1 protein. This
invention provides an antibody capable of specifically recognizing
MUM-1 protein. This invention provides a purified MUM-2 protein.
This invention provides an antibody directed to a purified MUM-2
protein. This invention provides an antibody capable of
specifically recognizing a MUM-2 protein.
[0018] This invention provides a pharmaceutical composition
comprising an amount of an oligonucleotide effective to prevent
overexpression of a human MUM-1 protein and a pharmaceutically
acceptable carrier capable of passing through a cell membrane. This
invention provides a pharmaceutical composition comprising an
amount of the oligonucleotide effective to prevent overexpression
of a human MUM-2 protein and a pharmaceutically acceptable carrier
capable of passing through a cell membrane.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1. JH-C.mu. dissociation in BamHI digested DNA of the
14q+ SK-MM-1 cell line. A 10 .mu.g of the high molecular weight DNA
was completely digested with BamHI, loaded on each lane and
blotted. The same filter was sequentially hybridized with JH,
C.mu., C.gamma.2, and 0.7B/H probes. JH probe detects two
rearranged bands of 12.0 kb and 9.7 kb. The 9.7 kb band is
comigrated with that probed with C.gamma.2 probe, suggesting it to
be a physiological rearrangement. On the other hand, one allele of
the C.mu.locus is deleted and another is rearranged (6.5 kb)
without being comigrated with rearranged bands of JH. Therefore,
12.0 kb and 6.5 kb bands detected by JH and C.mu. (shown by
arrowheads) might represent unknown derivative chromosome and
derivative 14 chromosome, respectively. As expected, 0.7B/H probe
(FIG. 2A) detected the rearranged band comigrated with 6.5 kb band
of C.mu.. Dashed lines show the comigration. Size markers of
.lamda./HindIII are shown on the left.
[0020] FIGS. 2A-B. Molecular cloning of the breakpoints of the
t(6;14) translocation and germline walking at MUM1 locus. (A)
Restriction maps of .lamda.SKB-4a and .lamda.SKS-3 clones
representing derivative 6 and 14 are shown, together with germline
maps of IgH locus at 14q32 and MUM1 locus at 6p25. Arrows indicate
the chromosomal breakpoints. B, BamHI; E, EcoRI; H, HindIII. (B)
Comparison of the nucleotide sequences around the breakpoints on
derivative 6 and derivative 14 chromosome. Homologous regions are
indicated by dashes. The arrow indicates the breakpoint. Nucleotide
numbers shown below are the same as in the Sp sequence reported by
Sun, et al. (18).
[0021] FIG. 3. Mapping of the MUM1 locus to chromosome 6p25.
.lamda.MUM-3 genomic clone (FIG. 2A) was used as a probe for in
situ hybridization. The white arrow indicates the fluorescence
signal on chromosome 6 band p25. Right panel shows the G-banding
picture stained with DAPI.
[0022] FIGS. 4A-C. Expression of the MUM1 gene in hematopoietic
lineage. A 10 ug aliquot of total RNA was loaded on each lane and
Northern blot analysis was performed using the 2.1H probe (FIG.
2A). GAPDH or .beta.-actin probes were used to control for amount
of RNA loaded. (A) MUM1 RNA expression in various hematopoietic
cell lines. MUM1 RNA is detected in B cell and mature T cell lines
as a single 6 kb transcript. HELA, epithelial lineage; LCL,
Epstein-Barr virus-transformed lymphoblastoid cell line; RAMOS and
SK-MM-1, B-cell lineage; HUT-78 and MOLT-4, T-cell lineage; HL-60
and U937, myelomonocytic lineage; K562, erythroid lineage. Dashes
indicate 28S and 18S. (B) Expression in B cell lines derived from
various stages of B cell differentiation. MUM1 RNA is seen
throughout the B cell development except for BJAB cell line. 697,
pre-B cell stage; RAMOS and BJA-B, Burkitt cell line representing
mature-B cell stage; RPMI-8226 and U-266, plasma cell stage. (C)
Comparison of the expression level among myeloma cell lines. MUM1
RNA is overexpressed in SK-MM-1 cell line carrying t(6;14).
Overexpression of the MUM1 is also demonstrated in XG-4, XG-7, and
XG-10 cell lines. RPMI-8226, U-266, EJM, and SKMM-1 are IL-6
(interleukin-6) independent lines, whereas XG-1, XG-2, XG-4, XG-5,
XG-6, XG-7, and XG-10 are IL-6 dependent lines.
[0023] FIGS. 5A-B. Sequence of MUM1 cDNA and structure of its
predicted protein product. (A) Restriction map of the MUM1 cDNA and
the position of the open reading frame (box). The solid box
indicates approximate position of the DNA binding domain. Sc,
SacII; A, ApaI; P, PstI; H, HindIII; S, SacI (B) Nucleotide
sequence of the MUM1 cDNA and corresponding amino acid sequence.
Putative translation initiation codons and preceding stop codons
appearing in frame are underlined. The asterisk indicates the
translation stop codon.
[0024] FIGS. 6A-B. Homology between MUM1 and other IRF family
proteins. (A) Similarity at N-terminal DNA binding domain. Black
background indicates identical residues found more than four times.
Gray indicates conserved residues that appear in at least four
sequences at a given position. Conserved tryptophan residues in DNA
binding domain among IRF family members are indicated by closed
circles. (B) Similarity at C-terminal region between human MUM1,
Mouse LSIRF/Pip, Human ICSBP, Human ISGF3.gamma., and Human IRF-3.
Black and gray background are as in (A).
[0025] FIG. 7. Genomic organization of the MUM1 gene and location
of the chromosomal breakpoints in multiple myeloma. Filled boxes
indicate the coding regions and empty boxes indicate the noncoding
regions. The position, and the size of each exon of the MUM1 gene
are approximate and have been determined by the hybridizations. One
exon in each restriction fragment may consist of more than two
exons. Translation initiation codon (ATG) and stop codon (TGA) are
indicated. Genomic probes used for further investigations are shown
as solid bars below the map. Arrows indicate the chromosomal
breakpoints of SKMM-l cell line and case 10. B, BamHI; E, EcoRI; H,
HindIII.
[0026] FIG. 8. Scheme of the t(6;14) (p25;q32) translocation
involving the MUM1 and the immunoglobulin heavy chain (IgH) gene
loci. VH-D-J-CH indicates variable-diversity joining-constant
region of the IgH gene. Direction of the MUM1 gene on the
chromosome 6 is tentatively drawn.
[0027] FIG. 9A-B. Demonstration of JH-Ca disjunction in U-266 cells
and cloning of normal and 14q+ chromosomal breakpoints. (A) The
panel shows the results of Southern blot analysis of BamHI digested
U-266 and normal control (placenta) genomic DNA using the indicated
JH and C.alpha. probes. The arrowheads indicate two DNA fragments
containing C.alpha. sequences not linked to JH sequences,
suggesting the presence of a chromosomal breakpoint in 14q32. (B)
The panel provides a schematic representation of the phage clones
isolated from a library constructed from U-266 DNA and screened
with a Ca probe. Based on restriction enzyme analysis, the three
cloned regions represent a normal Ca region (14q32 germ-line), and
two rearranged regions (der.14 and 14q32) containing unknown
sequences linked to C.alpha. sequences. The 2.5BE probe used for
Northern blot analysis of MUM2 transcripts (FIG. 10) is also
shown.
[0028] FIG. 10. Identification of MUM2 RNA transcripts. The figure
shows the results of a Northern blot analysis of RNA extracted from
various MM/PCL cell lines using the 2.5BE probe (see FIG. 9) or
GAPDH probe (as a control for RNA loading). A 1.9 Kb RNA transcript
is detectable in some cell lines including U-266, indicating that
the 2.5BE fragments represents part of a gene, MUM2.
[0029] FIG. 11A-B. Schematic representation of IgH DNA
rearrangements in normal B cells and in tumors carrying chromosomal
translocations breaking the S region of the IgH locus. Note that in
physiological IgH rearrangements (panel 11A) JH sequences and C
sequences (C.mu. before and C.gamma. after switch recombination,
respectively) are consistently found within the same BamHI
restriction fragment. Conversely, JH and C sequences are not
linked, and are present on two different chromosomes [derivative X
and derivative 14(14q+)] in cells carrying a chromosomal
translocation breaking the switch region (panel 11B)
[0030] FIG. 12A-B. MUM1 cDNA. cDNA insert is cloned into
EcoRI/BamHI site of the pBluescript KS+. Bacteria strain used is
DH5.alpha. cells. pcMUM1.16a contains full length open reading
frame of nt. 217-1572.
[0031] FIG. 13. Breakpoint Cloning of the U-266 Cell Line. pMUM2-8
has a 22.0 KB insert in BamHI site of pBluescript KS+.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The following standard abbreviations are used throughout the
specification to indicate specific nucleotides: [0033] C=cytosine
A=adenosine [0034] T=thymidine G=guanosine
[0035] This invention provides a method of determining a
chromosomal breakpoint in a subject suffering from multiple myeloma
which comprises steps of: (a) obtaining a DNA sample from the
subject suffering from multiple myeloma; (b) determining whether
there is J and C disjunction in the immunoglobulin heavy chain gene
in the obtained DNA sample; (c) obtaining a genomic library having
clones which contain genomic DNA fragments from the DNA sample
which shows positive J and C disjunction; (d) selecting and
isolating clones of the obtained library which show positive
hybridization with a probe which is capable of specifically
hybridizing with the C but not the J region of the immunoglobulin
heavy chain gene; (e) preparing fluorescent probes from the genomic
DNA fragments of the isolated clones from step (d); (f) hybridizing
said fluorescent probes with metaphase chromosomes; and (g)
determining the identity of the chromosomes which are capable of
hybridizing to said fluorescent probes, wherein the identification
of a chromosome other than chromosome 14 would indicate that the
chromosomal breakpoint is between chromosome 14 and the identified
chromosome, thereby determining a chromosomal breakpoint in a
subject suffering from multiple myeloma.
[0036] In an embodiment, step (b) of the above described method of
this invention is performed by Southern blotting. In another
embodiment, step (b) of the above method of this invention is
performed by polymerase chain reaction (PCR) with appropriate
probes. Polymerase chain reaction is well known in the art. Since
the sequences of both the C and J regions of an immunoglobulin
heavy chain gene are known, appropriate probes for PCR may
routinely be designed.
[0037] In an embodiment, the genomic library is a phage vector
library. In another embodiment, the genomic DNA fragments are
generated by cleaving genomic DNA from cells of the subject with an
appropriate restriction enzyme. In a further embodiment, the
restriction enzyme is BamHI. In an embodiment, the restriction
enzyme is Sau3AI. In another embodiment, the probe of step (d) is a
human IgH J region JH probe. In a further embodiment, the probe of
step (d) is a human IgH C.mu. probe. In an embodiment, the probe of
step (d) is a human IgH C.gamma.2 probe. In another embodiment, the
chromosomal breakpoint identified is a t(6;14) (p25;q32)
translocation. In an embodiment, the chromosomal breakpoint
identified is a t(14;15) translocation.
[0038] This invention provides a method to identify a gene other
than the immunoglobulin gene which is located in chromosome 14,
altered by a chromosomal breakpoint detected in a subject suffering
from multiple myeloma which comprises steps of: a) selecting a
probe having a sequence of a chromosome other than chromosome 14,
identified at the chromosomal breakpoint detected in a subject
suffering from multiple myeloma, wherein said probe is capable of
hybridizing to the unique sequence of the gene other than the
immunoglobulin gene altered by a chromosomal breakpoint detected in
a subject suffering from multiple myeloma; b) contacting said probe
with mRNA isolated from a cell under conditions permitting
formation of a complex between said probe and the mRNA; c)
isolating the complex resulting from step (b); and d) determining
the sequence of the mRNA in the isolated complex, thereby
determining the identity of the gene.
[0039] In an embodiment, step (d) of the method to identify a gene
other than the immunoglobulin gene which is located in chromosome
14, altered by a chromosomal breakpoint detected in a subject
suffering from multiple myeloma comprises steps of: i) synthesizing
complementary DNA to the mRNA; and ii) performing sequence analysis
of the complementary DNA to determine the sequence of the mRNA.
[0040] This invention provides a gene identified by the method to
identify a gene other than the immunoglobulin gene which is located
in chromosome 14, altered by a chromosomal breakpoint detected in a
subject suffering from multiple myeloma.
[0041] As used herein, "MUM" means any gene rearranged in
14q+chromosomal abnormalities associated with multiple myeloma.
[0042] This invention provides a gene identified by the above
method designated MUM-1. This invention provides a gene identified
by the above method designated MUM-2.
[0043] This invention provides a gene identified by the above
method, wherein the gene identified comprises a nucleic acid
encoding a MUM protein. In an embodiment, the gene identified by
the above method comprises a nucleic acid encoding a MUM-1 protein.
In another embodiment, the gene identified by the above method
comprises a nucleic acid encoding a MUM-2 protein.
[0044] This invention provides an isolated nucleic acid molecule
encoding a MUM protein. In an embodiment, the isolated nucleic acid
molecule encoding a MUM protein is a DNA molecule. In another
embodiment, the isolated nucleic acid molecule encoding a MUM
protein is a cDNA molecule.
[0045] In an embodiment, a cDNA nucleic acid molecule encoding a
MUM-1 protein is cloned into a pBluescript KS+ and the resulting
plasmid is designated as pcMUM1-1.6a (ATCC Accession No. ______).
Plasmid pcMUM1-1.6a was deposited on May 28, 1996 with the American
Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville,
Md. 20852, U.S.A. under the provisions of the Budapest Treaty for
the International Recognition of the Deposit of Microorganisms for
the Purposes of Patent Procedure. Plasmid pcMUM1-1.6a was accorded
ATCC Accession No. ______.
[0046] In another embodiment, a partial cDNA nucleic acid molecule
encoding a MUM-1 protein is cloned into a pBluescript KS+ and the
resulting plasmid is designated as pMUM1-2.4B/N (ATCC Accession No.
______). Plasmid pMUM1-2.4B/N was deposited on May 28, 1996 with
the American Type Culture Collection (ATCC), 12301 Parklawn Drive,
Rockville, Md. 20852, U.S.A. under the provisions of the Budapest
Treaty for the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure. Plasmid
pMUM1-2.4B/N was accorded ATCC Accession No. ______.
[0047] In another embodiment, a partial cDNA nucleic acid molecule
encoding a MUM-1 protein is cloned into a pBluescript KS+ and the
resulting plasmid is designated as pMUM1-7.7B (ATCC Accession No.
______). Plasmid pMUM1-7.7B was deposited on May 28, 1996 with the
American Type Culture Collection (ATCC), 12301 Parklawn Drive,
Rockville, Md. 20852, U.S.A. under the provisions of the Budapest
Treaty for the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure. Plasmid
pMUM1-7.7B was accorded ATCC Accession No. ______.
[0048] In another embodiment, a partial cDNA of the nucleic acid
molecule encoding a MUM-2 protein is cloned into a pBluescript KS+
and the resulting plasmid is designated as pMUM2-8 (ATCC Accession
No. ______). Plasmid pMUM2-8 was deposited on May 28, 1996 with the
American Type Culture Collection (ATCC), 12301 Parklawn Drive,
Rockville, Md. 20852, U.S.A. under the provisions of the Budapest
Treaty for the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure. Plasmid
pMUM2-8 was accorded ATCC Accession No. ______.
[0049] In an embodiment, the isolated DNA molecule encoding a MUM
protein is a cDNA molecule having the nucleotide sequence shown in
FIG. 5B (SEQ. ID NO ).
[0050] In an embodiment, the isolated DNA molecule encoding a MUM
protein is genomic DNA molecule. In an embodiment, the isolated
nucleic acid molecule encoding a MUM protein is an RNA
molecule.
[0051] In an embodiment, the isolated nucleic acid encodes a human
MUM-1 protein. In another embodiment, the isolated nucleic acid
molecule encodes a human MUM-2 protein.
[0052] In an embodiment, isolated nucleic molecule encodes the a
human MUM-1 protein having substantially the same amino acid
sequence as shown in FIG. 5B (SEQ. ID NO ). In an embodiment,
isolated nucleic molecule encodes a human MUM-1 protein having the
same amino acid sequence as shown in FIG. 5B (SEQ. ID NO ). In an
embodiment, the isolated nucleic acid molecule encoding a MUM
protein is operatively linked to a promoter of RNA
transcription.
[0053] This invention provides a vector comprising a cDNA molecule
encoding a MUM protein. In an embodiment, a vector comprising cDNA
encoding for MUM-1 is designated pcMUM1.6a. In an embodiment, a
vector comprising partial cDNA encoding for MUM-1 is designated
pMUM1.2.4B/N. In an embodiment, a vector comprising partial cDNA
encoding for MUM-1 is designated pMUM1-7.7B. In an embodiment, a
vector comprising partial cDNA encoding for MUM-2 is designated
pMUM2-8. In an embodiment, a vector comprises genomic DNA encoding
for MUM. In an embodiment, the vector is a plasmid. In an
embodiment, a host cell comprises the vector comprising cDNA
encoding for MUM. In an embodiment, a host cell comprises the
vector comprising genomic DNA encoding for MUM. In a further
embodiment, the host cell comprising vectors comprising cDNA
encoding for MUM or comprising genomic DNA encoding for MUM is
selected from a group consisting of a bacterial cell, a plant cell,
and insect cell and a mammalian cell.
[0054] This invention provides a nucleic acid probe comprising a
nucleic acid molecule of at least 15 nucleotides capable of
specifically hybridizing with a unique sequence included within the
sequence of a nucleic acid molecule encoding a MUM protein. This
invention provides a nucleic acid probe comprising a nucleic acid
molecule of at least 15 nucleotides which is complementary to a
sequence of the isolated nucleic acid molecule encoding a MUM
protein.
[0055] As used herein, the phrase "specifically hybridizing" means
the ability of a nucleic acid molecule to recognize a nucleic acid
sequence complementary to its own and to form double-helical
segments through hydrogen bonding between complementary base
pairs.
[0056] In an embodiment, the nucleic acid probe specifically
hybridizes with nucleic acid encoding MUM-1. In an embodiment, the
nucleic acid probe is complementary to nucleic acid encoding MUM-1.
In an embodiment, the nucleic acid probe specifically hybridizes
with nucleic acid encoding MUM-2. In an embodiment, the nucleic
acid probe is complementary to nucleic acid encoding MUM-2.
[0057] In an embodiment, the nucleic acid probe which specifically
hybridizes with nucleic acid encoding MUM-1 is a DNA probe. In an
embodiment, the nucleic acid probe which specifically hybridizes
with nucleic acid encoding MUM-2 is a DNA probe.
[0058] In an embodiment, the nucleic acid probe which specifically
hybridizes with nucleic acid encoding MUM-1 is a RNA probe. In an
embodiment, the nucleic acid probe which specifically hybridizes
with nucleic acid encoding MUM-2 is a RNA probe.
[0059] In an embodiment, the nucleic acid probe which specifically
hybridizes with nucleic acid encoding MUM-1 is a genomic DNA probe.
In an embodiment, the nucleic acid probe which specifically
hybridizes with nucleic acid encoding MUM-2 is a genomic DNA
probe.
[0060] In an embodiment, the nucleic acid probe which specifically
hybridizes with nucleic acid encoding MUM-1 is labeled with a
detectable marker. In an embodiment, the nucleic acid probe which
specifically hybridizes with nucleic acid encoding MUM-2 is labeled
with a detectable marker.
[0061] In an embodiment, the detectable marker is selected from the
group consisting of a radioactive isotope, enzyme, dye, biotin, a
fluorescent label or a chemiluminescent label.
[0062] In an embodiment, the nucleic acid probe which specifically
hybridizes with nucleic acid encoding MUM-1 is linked to a nucleic
acid sequence capable of specifically hybridizing with a unique
sequence included within the sequence of a nucleic acid molecule of
human chromosome 14. In an embodiment, the nucleic acid probe which
specifically hybridizes with nucleic acid encoding MUM-2 is linked
to a nucleic acid sequence capable of specifically hybridizing with
a unique sequence included within the sequence of a nucleic acid
molecule of human chromosome 14.
[0063] This invention provides a nucleic acid probe comprising a
nucleic acid molecule of at least 15 nucleotides which is
complementary to a sequence of the isolated nucleic acid molecule
encoding a MUM protein which is linked to a nucleic acid sequence
complementary to a sequence of a nucleic acid molecule of human
chromosome 14.
[0064] In an embodiment, the nucleic acid probe comprises a nucleic
acid molecule of at least 15 nucleotides which is complementary to
a sequence of the isolated nucleic acid molecule encoding a MUM-1
protein which is linked to a nucleic acid sequence complementary to
a sequence of a nucleic acid molecule of human chromosome 14.
[0065] In an embodiment, the nucleic acid probe comprises a nucleic
acid molecule of at least 15 nucleotides which is complementary to
a sequence of the isolated nucleic acid molecule encoding a MUM-2
protein which is linked to a nucleic acid sequence complementary to
a sequence of a nucleic acid molecule of human chromosome 14.
[0066] In an embodiment, the nucleic acid probe comprises a nucleic
acid molecule of at least 15 nucleotides which is complementary to
a sequence of the isolated nucleic acid molecule encoding a MUM-1
protein which is linked at a specific break point to a nucleic acid
sequence complementary to a sequence of a nucleic acid molecule of
human chromosome 14.
[0067] In an embodiment, the nucleic acid probe comprises a nucleic
acid molecule of at least 15 nucleotides which is complementary to
a sequence of the isolated nucleic acid molecule encoding a MUM-2
protein which is linked at a specific break point to a nucleic acid
sequence complementary to a sequence of a nucleic acid molecule of
human chromosome 14.
[0068] In an embodiment, the specific break point of the nucleic
acid probe comprises a portion of the t(6;14) (p25;q32)
translocation. In an embodiment, the specific break point of the
nucleic acid probe comprises a portion of a t(14;15) translocation.
In an embodiment, the nucleic acid probe comprising a portion of
the t(6;14)(p25;q32) translocation is labeled with a detectable
marker. In an embodiment, the nucleic acid probe comprising a
portion of a t(14;15) translocation is labeled with a detectable
marker. In an embodiment, the nucleic acid probe comprising a
portion of the t(6;14)(p25;q32) or comprising a portion of a
t(14;15) translocation of claim 60, has a detectable marker
selected from the group consisting of a radioactive isotope,
enzyme, dye, biotin, a fluorescent label or a chemiluminescent
label.
[0069] This invention provides a method for detecting a
predisposition to multiple myeloma associated with the expression
of a human MUM-1 protein in a sample from a subject which comprises
detecting in a sample from the subject a rearrangement of nucleic
acid encoding MUM-1 protein.
[0070] This invention provides a method for detecting a
predisposition to multiple myeloma associated with the expression
of a human MUM-2 protein in a sample from a subject which comprises
detecting in a sample from the subject a rearrangement of nucleic
acid encoding MUM-2 protein.
[0071] In an embodiment, the rearrangement of nucleic acid encoding
MUM-1 protein is detected by contacting the nucleic acid from the
sample with a MUM-1 probe under conditions permitting the MUM-1
probe to hybridize with the nucleic acid encoding MUM-1 protein
from the sample, thereby detecting the rearrangement of nucleic
acid encoding MUM-1 protein in the sample.
[0072] In an embodiment, the rearrangement of nucleic acid encoding
MUM-2 protein is detected by contacting the nucleic acid from the
sample with a MUM-2 probe under conditions permitting the MUM-2
probe to hybridize with the nucleic acid encoding MUM-2 protein
from the sample, thereby detecting the rearrangement of nucleic
acid encoding MUM-2 protein in the sample.
[0073] In an embodiment, the rearrangement of nucleic acid encoding
MUM-1 protein is detected by a MUM-1 probe comprising a nucleic
acid molecule of at least 15 nucleotides which is complementary to
a sequence of the isolated nucleic acid molecule encoding MUM-1
protein which is linked to a nucleic acid sequence complementary to
a sequence of a nucleic acid molecule of human chromosome 14.
[0074] In an embodiment, the rearrangement of nucleic acid encoding
MUM-2 protein is detected by a the MUM-2 probe comprising a nucleic
acid molecule of at least 15 nucleotides which is complementary to
a sequence of the isolated nucleic acid molecule encoding MUM-2
protein which is linked to a nucleic acid sequence complementary to
a sequence of a nucleic acid molecule of human chromosome 15.
[0075] In an embodiment, the MUM-1 probe comprising a nucleic acid
molecule of at least 15 nucleotides which is complementary to a
sequence of the isolated nucleic acid molecule encoding MUM-1
protein is linked at a specific break point to a nucleic acid
sequence complementary to a sequence of a nucleic acid molecule of
human chromosome 14.
[0076] In an embodiment, the MUM-2 probe comprising a nucleic acid
molecule of at least 15 nucleotides which is complementary to a
sequence of the isolated nucleic acid molecule encoding MUM-2
protein is linked at a specific break point to a nucleic acid
sequence complementary to a sequence of a nucleic acid molecule of
human chromosome 15.
[0077] In an embodiment, the MUM-1 probe comprises a specific break
point comprising a portion of the t(6;14)(p25;q32) translocation.
In an embodiment, the MUM-2 probe comprises a specific break point
comprising a portion of a t(14;15) translocation.
[0078] In an embodiment, the method for detecting a predisposition
to multiple myeloma associated with the expression of a human MUM-1
protein in a sample from a subject which comprises detecting in a
sample from the subject a rearrangement of nucleic acid encoding
MUM-1 protein comprises: a) obtaining DNA from the sample of the
subject suffering from multiple myeloma; b) performing a
restriction digest of the DNA with a panel of restriction enzymes;
c) separating the resulting DNA fragments by size fractionation; d)
contacting the resulting DNA fragments with a nucleic acid probe
capable of specifically hybridizing with a unique sequence included
within the sequence of a nucleic acid molecule encoding a human
MUM-1 protein, wherein the sequence of a nucleic acid molecule
encoding a MUM-1 protein is linked at a specific break point to a
specified nucleic acid sequence of human chromosome 14 and labeled
with a detectable marker; e) detecting labeled bands which have
hybridized to the nucleic acid probe capable of specifically
hybridizing with a unique sequence included within the sequence of
a nucleic acid molecule encoding a human MUM-1 protein, wherein the
sequence of a nucleic acid molecule encoding a MUM-1 protein is
linked at a specific break point to a specified nucleic acid
sequence of human chromosome 14 to create a unique band pattern
specific to the DNA of subjects suffering from multiple myeloma; f)
preparing DNA obtained from a sample of a subject for diagnosis by
steps (a-e); and g) comparing the detected band pattern specific to
the DNA obtained from a sample of subjects suffering from multiple
myeloma from step (e) and the DNA obtained from a sample of the
subject for diagnosis from step (f) to determine whether the
patterns are the same or different and to diagnose thereby
predisposition to multiple myeloma if the patterns are the
same.
[0079] In an embodiment, the method for detecting a predisposition
to multiple myeloma associated with the expression of a human MUM-2
protein in a sample from a subject which comprises detecting in a
sample from the subject a rearrangement of nucleic acid encoding
MUM-2 protein comprises: a) obtaining DNA from the sample of the
subject suffering from multiple myeloma; b) performing a
restriction digest of the DNA with a panel of restriction enzymes;
c) separating the resulting DNA fragments by size fractionation; d)
contacting the resulting DNA fragments with a nucleic acid probe
capable of specifically hybridizing with a unique sequence included
within the sequence of a nucleic acid molecule encoding a human
MUM-2 protein, wherein the sequence of a nucleic acid molecule
encoding a MUM-2 protein is linked at a specific break point to a
specified nucleic acid sequence of human chromosome 14 and labeled
with a detectable marker; e) detecting labeled bands which have
hybridized to the nucleic acid probe capable of specifically
hybridizing with a unique sequence included within the sequence of
a nucleic acid molecule encoding a human MUM-2 protein, wherein the
sequence of a nucleic acid molecule encoding a MUM-2 protein is
linked at a specific break point to a specified nucleic acid
sequence of human chromosome 14 to create a unique band pattern
specific to the DNA of subjects suffering from multiple myeloma; f)
preparing DNA obtained from a sample of a subject for diagnosis by
steps (a-e); and g) comparing the detected band pattern specific to
the DNA obtained from a sample of subjects suffering from multiple
myeloma from step (e) and the DNA obtained from a sample of the
subject for diagnosis from step (f) to determine whether the
patterns are the same or different and to diagnose thereby
predisposition to multiple myeloma if the patterns are the
same.
[0080] In an embodiment, the size fractionation in step (c) is
effected by a polyacrylamide or agarose gel. In an embodiment, the
detectable marker is radioactive isotope, enzyme, dye, biotin, a
fluorescent label or a chemiluminescent label.
[0081] In an embodiment, the method for detecting a predisposition
to multiple myeloma associated with the expression of a human MUM-1
protein in a sample from a subject which comprises detecting in a
sample from the subject a rearrangement of nucleic acid encoding
MUM-1 protein comprises: a) obtaining RNA from the sample of the
subject suffering from multiple myeloma; b) separating the RNA
sample by size fractionation; c) contacting the resulting RNA
species with a nucleic acid probe capable of specifically
hybridizing with a unique sequence included within the sequence of
a nucleic acid molecule encoding a human MUM-1 protein, wherein the
sequence of a nucleic acid molecule encoding a MUM-1 protein is
linked at a specific break point to a specified nucleic acid
sequence of human chromosome 14 and labeled with a detectable
marker; d) detecting labeled bands which have hybridized to the RNA
species to create a unique band pattern specific to the RNA of
subjects suffering from multiple myeloma; e) preparing RNA obtained
from a sample of a subject for diagnosis by steps (a-d); and f)
comparing the detected band pattern specific to the RNA obtained
from a sample of subjects suffering from multiple myeloma from step
(d) and the RNA obtained from a sample of the subject for diagnosis
from step (f) to determine whether the patterns are the same or
different and to diagnose thereby predisposition to multiple
myeloma if the patterns are the same.
[0082] In an embodiment, the method for detecting a predisposition
to multiple myeloma associated with the expression of a human MUM-2
protein in a sample from a subject which comprises detecting in a
sample from the subject a rearrangement of nucleic acid encoding
MUM-2 protein comprises: a) obtaining RNA from the sample of the
subject suffering from multiple myeloma; b) separating the RNA
sample by size fractionation; c) contacting the resulting RNA
species with a nucleic acid probe capable of specifically
hybridizing with a unique sequence included within the sequence of
a nucleic acid molecule encoding a human MUM-2 protein, wherein the
sequence of a nucleic acid molecule encoding a MUM-2 protein is
linked at a specific break point to a specified nucleic acid
sequence of human chromosome 15 and labeled with a detectable
marker; d) detecting labeled bands which have hybridized to the RNA
species to create a unique band pattern specific to the RNA of
subjects suffering from multiple myeloma; e) preparing RNA obtained
from a sample of a subject for diagnosis by steps (a-d); and f)
comparing the detected band pattern specific to the RNA obtained
from a sample of subjects suffering from multiple myeloma from step
(d) and the RNA obtained from a sample of the subject for diagnosis
from step (f) to determine whether the patterns are the same or
different and to diagnose thereby predisposition to multiple
myeloma if the patterns are the same.
[0083] In an embodiment, the size fractionation in step (b) is
effected by a polyacrylamide or agarose gel. In an embodiment, the
detectable marker is radioactive isotope, enzyme, dye, biotin, a
fluorescent label or a chemiluminescent label.
[0084] In an embodiment, multiple myeloma associated with the
expression of a specific human MUM-1 is diagnosed by the method for
detecting a predisposition to multiple myeloma associated with the
expression of a human MUM-1 protein in a DNA or RNA sample from a
subject which comprises detecting in a sample from the subject a
rearrangement of nucleic acid encoding MUM-1 protein.
[0085] In an embodiment, multiple myeloma associated with the
expression of a specific human MUM-2 is diagnosed by the method for
detecting a predisposition to multiple myeloma associated with the
expression of a human MUM-12 protein in a DNA or RNA sample from a
subject which comprises detecting in a sample from the subject a
rearrangement of nucleic acid encoding MUM-2 protein.
[0086] This invention provides an antisense oligonucleotide having
a sequence capable of specifically hybridizing to an mRNA molecule
encoding a human MUM-1 protein so as to prevent overexpression of
the mRNA molecule. This invention provides an antisense
oligonucleotide having a sequence capable of specifically
hybridizing to an mRNA molecule encoding a human MUM-2 protein so
as to prevent overexpression of the mRNA molecule.
[0087] This invention provides an antisense oligonucleotide having
a sequence capable of specifically hybridizing to the cDNA molecule
encoding a MUM protein. This invention provides an antisense
oligonucleotide having a sequence capable of specifically
hybridizing to the genomic DNA molecule encoding a MUM protein.
This invention provides an antisense oligonucleotide having a
sequence capable of specifically hybridizing to the RNA molecule
encoding a MUM protein.
[0088] This invention provides a purified MUM protein. This
invention provides a purified MUM-1 protein. This invention
provides a purified human MUM-1 protein. This invention provides an
antibody directed to a purified MUM-1 protein. This invention
provides an antibody capable of specifically recognizing MUM-1
protein. In an embodiment, the antibody capable of specifically
recognizing MUM-1 protein is a human MUM-1 protein.
[0089] This invention provides a purified MUM-2 protein. This
invention provides a purified human MUM-2 protein. This invention
provides an antibody directed to a purified MUM-2 protein. This
invention provides an antibody capable of specifically recognizing
MUM-2 protein. In an embodiment, the antibody capable of
specifically recognizing MUM-2 protein is a human MUM-2
protein.
[0090] In an embodiment, the antibody directed to a purified MUM-1
protein is a monoclonal antibody. In an embodiment, the antibody
capable of specifically recognizing MUM-1 protein is a monoclonal
antibody. In an embodiment, the antibody capable of specifically
recognizing MUM-1 protein is a human MUM-1 protein.
[0091] In an embodiment, the antibody directed to a purified MUM-2
protein is a monoclonal antibody. In an embodiment, the antibody
capable of specifically recognizing MUM-2 protein is a monoclonal
antibody. In an embodiment, the antibody capable of specifically
recognizing MUM-2 protein is a human MUM-2 protein.
[0092] This invention provides a pharmaceutical composition
comprising an amount of the oligonucleotide having a sequence
capable of specifically hybridizing to an mRNA molecule encoding a
human MUM-1 protein so as to prevent overexpression of the mRNA
molecule effective to prevent overexpression of a human MUM-1
protein and a pharmaceutically acceptable carrier capable of
passing through a cell membrane.
[0093] This invention provides a pharmaceutical composition
comprising an amount of the oligonucleotide having a sequence
capable of specifically hybridizing to a cDNA molecule encoding a
MUM protein effective to prevent overexpression of a human MUM-1
protein and a pharmaceutically acceptable carrier capable of
passing through a cell membrane.
[0094] This invention provides a pharmaceutical composition
comprising an amount of the oligonucleotide having a sequence
capable of specifically hybridizing to a genomic DNA molecule
effective to prevent overexpression of a human MUM-1 protein and a
pharmaceutically acceptable carrier capable of passing through a
cell membrane.
[0095] This invention provides a pharmaceutical composition
comprising an amount of the oligonucleotide having a sequence
capable of specifically hybridizing to an mRNA molecule encoding a
human MUM-2 protein so as to prevent overexpression of the mRNA
molecule effective to prevent overexpression of a human MUM-2
protein and a pharmaceutically acceptable carrier capable of
passing through a cell membrane.
[0096] This invention provides a pharmaceutical composition
comprising an amount of the oligonucleotide having a sequence
capable of specifically hybridizing to a cDNA molecule encoding a
MUM protein effective to prevent overexpression of a human MUM-2
protein and a pharmaceutically acceptable carrier capable of
passing through a cell membrane.
[0097] This invention provides a pharmaceutical composition
comprising an amount of the oligonucleotide having a sequence
capable of specifically hybridizing to a genomic DNA molecule
effective to prevent overexpression of a human MUM-2 protein and a
pharmaceutically acceptable carrier capable of passing through a
cell membrane.
[0098] This invention will be better understood from the
Experimental Details which follow. However, one skilled in the art
will readily appreciate that the specific methods and results
discussed are merely illustrative of the invention as described
more fully in the claims which follow thereafter.
Experimental Details
Materials and Methods
[0099] Cell lines. The following myeloma cell lines were used in
the present study: SK-MM-1, RPMI-8226, U266, EJM, XG-1, XG-2, XG-4,
XG-5, XG-6, XG-7, and XG-10. The RPMI-8226 cell line was obtained
through the American Type Culture Collection (ATCC, Rockville,
Md.). SKMM-1 and U-266 cell lines were gifts from Dr. A. N.
Houghton and Dr. K. Nilsson, respectively (18; 12).
Characterization of these cell lines were previously reported. Six
XG cell lines were gifts from Dr. B. Klein and were cultured in
RPMI 1640 containing 10% fetal calf serum (FCS), SxlO-smol/L 2-ME,
and rIL-6(1 ng/mL)(13;19). Other myeloma cell lines used were all
IL-6 independent. The SK-MM-l cell line was used to isolate the
chromosomal breakpoint carrying the 14q+chromosome without any
information on the donor chromosome. XG-1, XG-2, XG-6, XG-8 cell
lines are reported to carry the t(11;14) (ql3;q32) translocation.
XG-5 cells also share both t(11;14) and t(8;14) (q24;q32).
[0100] Southern and Northern blot analyses. Southern blot analysis
was performed as previously described (21). Briefly, ten micrograms
of high molecular-weight DNA extracted from each cell line was
digested to completion with BamHI and HindIII restriction enzymes,
size-fractionated on 0.7% agarose gel, and transferred onto
Duralose nitrocellulose membrane (Stratagene) according to the
manufacturer's instructions. Blots were hybridized with a
random-primed DNA probe and washed at 60.degree. C. in
0.2.times.SSC and 0.1% SDS for 5 minutes. Genomic probes used in
this study were as follows; human IgH J region JH probe (6.6 kb
BamHI-HindIII fragment) was provided by Dr. J. V. Ravetch, human
IgH C.mu. probe (1.3 kb EcoRI fragment) was provided by Dr. S. J.
Korsmeyer. Human IgH region C.gamma.2 probe was provided by Dr. C.
Croce.
[0101] Northern blot analysis was performed as described previously
(21). Briefly, a 10 .mu.g aliquot of total RNA was loaded on each
lane and probed with a 2.1H probe of the MUMI gene (FIG. 2A). GAPDH
or .beta.-actin probes were used as controls for amount of total
RNA.
[0102] Genomic library. High molecular-weight DNA of SK-MM-1 cell
line was digested completely with BamHI and partially with Sau3AI,
and size-fractionated by using a low-melting point agarose gel. DNA
ranging from 10 kb to 23 kb were purified and ligated into the
BamHI sites of .lamda.-DASH II phage vector (Stratagene, La Jolla,
Calif.). After packaging, 3.times.10.sup.5 and 6.times.10.sup.5
recombinant clones of the BamHI digested library and partially
digested library were screened with JH and C.mu. probes,
respectively. To isolate the germline region of the 6p25 locus, a
commercially available human placental library (Stratagene) was
screened. Positive clones were mapped with restriction enzymes by
partial digestion of the phage DNAs followed by probing with T7 and
T3 primers labeled with T4 polynucleokinase and 32p-.gamma.ATP.
[0103] cDNA library. A phage library constructed by oligo-dT and
random-priming normal human spleen RNA (Clontech) was screened by
2.1H probe (FIG. 2A) to isolate initial MUM1 cDNA clones. After the
first round of screening, positive clones were used as probes to
walk to the 5' side using the same library. Positive clones were
subcloned into pBluescript and analyzed for mapping and
sequencing.
[0104] DNA sequencing. DNA sequences were determined by the dideoxy
chain termination method and analyzed by an ABI(Applied Biosystems)
autosequencer. Deletion mutants for sequencing were prepared using
exonuclease III and mung bean nuclease. cDNA sequences were
analyzed with the Genetics Computer Group (GCG) programs. Sequence
homology searches were carried out through the BLAST E-mail server
at the National Center for Biotechnology Information, National
Library of Medicine, Bethesda, Md.
[0105] Fluorescence in situ hybridization (FISH). Metaphase
chromosome from human lymphocytes were prepared. A biotin-labeled
probe was prepared by nick-translation using Bio-16-dUTP.
Conditions for hybridization and washing were described previously
(22).
Experimental Results
[0106] IgH gene rearrangement of the SK-MM-I cell line. In BamHI
digestion, the JH probe detects two rearranged bands of the size of
12.0 kb and 9.7 kb (FIG. 1). The 9.7 kb band is comigrated with
that probed with C.gamma.2 probe, suggesting it to be a
physiological rearrangement, although this cell line secretes only
.lamda. chain. One allele of the C.mu. locus is deleted and another
is rearranged (6.5 kb) without being comigrated with rearranged
bands of JH. Hybridization with a C.alpha. probe showed only the
germline band (data not shown). These results suggested the
possibility of the chromosomal breakpoint between JH and C.mu.
locus. Hence, the 12.0 kb and the 6.5 kb bands detected by JH and
C.mu. were considered to represent unknown derivative chromosome
and derivative 14 chromosome, respectively.
[0107] Molecular cloning of the t(6;14) (p25;q32) breakpoint. A
genomic library constructed with BamHI complete digestion was
screened with a JH probe to isolate the 12.0 kb BamHI band. Another
library constructed with Sau3AI partial digestion was screened with
a C.mu. probe to isolate phage clones containing the 6.5 kb BamHI
fragment. Two phage clones, .lamda.SKB-4a and .lamda. SKS-3,
considered to represent the unknown derivative and derivative 14
chromosomes respectively, were obtained (FIG. 2A). A 0.7 kb
BamHI-HindIII probe (0.7B/H) of the .lamda.SKS-3 was used to
confirm the comigration with the rearranged 6.5 kb C.mu. band by
Southern analysis (FIG. 1). The chromosomal origin of the
centromeric side of the .lamda.SKB-4a and telomeric side of the
.lamda.SKS-3 were confirmed by hybridization to a somatic cell
hybrid DNA panel with a 4.5 kb ApaI fragment(4.5A) and 2.1 kb
HindIII(2.1H) probes. Both probes showed positive signals in hybrid
cell DNA containing a human chromosome 6 (data not shown). These
probes were also used to isolate the germline chromosome 6 region
by screening the human placental genomic library. One of the phage
clone DNA (.lamda.MUM-3) was used as a probe for FISH analysis. It
identified the localization of this region to be chromosome 6 short
arm p25 (FIG. 3). To investigate the precise breakpoint within the
IgH gene, a 1.5 kb HindIII-EcoRI fragment of the .lamda.SKS-3,
containing the breakpoint on derivative 14 chromosome was
sequenced. The breakpoint was confirmed to be just 3' to the switch
.mu.(S .mu.) repetitive sequences (FIG. 2B). Nucleotide sequencing
of the region around the breakpoints of chromosome 6 and derivative
6 chromosome showed that the chromosomal translocation was
reciprocal with minimum deletion of both the IgH and 6p25
sequences.
Transcriptional Unit in the Vicinity of the 6p25 Breakpoint.
[0108] An attempt to find a functional transcriptional unit in the
vicinity of the breakpoints was made. Although a 4.5A probe on
derivative 6 chromosome could not detect any transcripts, a 2.1H
probe on derivative 14 chromosome detected a single 6 kb transcript
in the SK-MM-1 cell line. Accordingly, this gene was designated as
MUM1 (multiple myeloma oncogene 1). The same probe was used to
study the expression of the MUM1 gene in various hematopoietic cell
lines. The 6 kb message was expressed at high levels in most B cell
lines and at low levels in peripheral T cell lines (FIG. 4A). Cell
lines derived from immature T cells, the myelomonocytic lineage,
and erythroid lineage do not seem to express MUM1. In B cells, MUM1
appears to be expressed throughout the development from the preB
cell stage to the plasma cell stage (FIG. 4B). However, some of the
Burkitt's lymphoma derived cell lines such as BJA-B did not express
this gene (data not shown). The expression level of the MUM1
transcript in myeloma cell lines was also examined (FIG. 4C). The
SK-MM-1 cell line showed a 7.5-fold overexpression when compared
with the other three IL-6 independent cell lines, suggesting a
deregulated expression of the translocated allele. It is of
interest that the IL-6 dependent XG-4, XG-7, and XG-10 cell lines
are also expressing at high levels. Particularly, expression in the
XG-7 cell line is 19.9 times the average of the aforementioned
control cell lines.
MUMI cDNA Cloning, Sequencing, and Homology Search.
[0109] Human spleen cDNA library was initially screened with a 2.1H
probe followed by three times walking to 5' side using cDNA probes.
A 5.5 kb cDNA, approximately corresponding to the size detected by
Northern analysis was isolated. This cDNA contained a 1,353 base
pair open reading frame (ORF) and a long 3' untranslated region
(FIG. 5A). The ORF encodes for a protein of 451 amino acids with a
predicted molecular weight of 50 kD (FIG. 5B). The putative ATG
initiation codon at position 217 has G at the -3 position which
corresponds to the Kozak consensus sequence (23). The ORF is
preceded by two in-frame stop codons. A database search
demonstrated a significant similarity between MUM-1 ORF and the
interferon regulatory factor (IRF) family proteins. The
NH.sub.2-terminal of the MUM-1 ORF shares a high homology with all
of the IRF family proteins which share a characteristic DNA binding
motif consisting of the conserved 5 tryptophan residues (FIG. 6A).
The COOH-terminal also has a high homology with ICSBP (interferon
consensus sequence binding protein) (21), ISGF3.gamma.
(interferon-stimulated gene factor-3 gamma) (22), and IRF-3 protein
(23) (FIG. 6B), although it did not have any homologous regions
with IRF-1 and IRF-2 protein. The highest similarity (95.1%) and
identity (91.8%) were found with a possible mouse homolog, LSIRF
(lymphoid specific interferon regulatory factor)/Pip (PU-1 cofactor
protein-1)(24,25). A high similarity was found with ICSBP (63.98%),
ISGF3.gamma. (55.8%), and IRF3 (50.1%) among the human IRF family
protein members. A gene sequence encoding a nearly identical
protein was recently deposited in GenBank. This gene, termed ICSAT
(interferon consensus sequence binding protein in adult T-cell
leukemia cell lines or activated T cells) is likely to be the same
gene as MUM1 (26).
Breakpoints at MUM1 Locus in Multiple Myeloma.
[0110] In order to analyze the exact location of the SK-MM-1
breakpoint at the 6p25 locus and to explore the frequency of the
MUM1 gene involvement in myeloma cases, we walked nearly 55 kb in a
human placental genomic phage library around the MUM1 gene and
determined the rough exon-intron structure as shown in FIG. 7 (FIG.
7). The SK-MM-1 breakpoint was located 3' to the last exon,
containing a poly A additional signal, consistent with an unaltered
size of the MUM1 transcript of this cell line in Northern analysis.
Seven repeat-free genomic probes shown in FIG. 7 have been used to
investigate the rearrangement in Southern analyses of the 11 MM
cell lines and 18 MM cases. One case (case 10) displayed rearranged
bands in BamHI and XbaI digests when analyzed using a 0.9A probe
located at 3' to the MUM1 gene.
Cloning of the MUM2 Locus from the U-266 Multiple Myeloma Cell
Line.
[0111] Using an experimental strategy analogous to the one
described for the cloning of the MUM1 gene from the SK-MM-1 cell
line, a second genetic locus altered in multiple myeloma (MUM2) was
identified by analyzing the U-266 multiple myeloma cell line.
Briefly, Southern blot analysis using BamH restiction digestion and
various Ig probes showed that U-266 DNA contained two rearranged
fragments (shown by arrowheads in FIG. 9) containing C.alpha.
sequences and lacking J sequences. These two fragments (der 14 and
14q32 in FIG. 9) were cloned from a genomic library constructed
from U-266 DNA along with a normal 14q 32 locus (14q32 germline in
FIG. 9). In order to determine whether a gene was located in
proximity to the chromosomal breakpoints in der 14, the 2.5 BE
restriction fragment (see FIG. 9), which was at the opposite side
of the Ig Ca sequences, was used to probe a Northern blot carrying
RNA from various MM cell lines. The results (FIG. 10) showed that a
1.9 kb mRNA was detectable in some of these cell lines including
U-266. This result showed that a gene, called MUM2, normally not
present within the Ig locus on chromosome 14q32, had been
translocated in proximity of the Ig locus in U-266 cells. Since the
Ig locus contains strong transcriptional regulatory elements, it is
likely that the expression of this gene is deregulated in these
cells. The structure of the MUM2 gene and its protein are currently
under investigation. The 2.5 BE probe and other probes derived from
the der 14 phage can be used to screen MM cases for MUM2
rearrangements as shown for MUM1 (FIG. 7).
Experimental Discussion
[0112] Using the experimental strategies used for the
identification of the MUM1 and MUM2 genes in the SK-MM-1 and U-266
cell lines, respectively, it is possible to analyze most MM cases
and isolate the corresponding genes. The scheme shown in FIG. 11
shows that the physiological IgH gene rearrangements (FIG. 11A)
typically maintain linkage of C and J sequences and this linkage
becomes detectable by using an appropriate restriction enzyme
digestion (BamHI in the example in FIG. 11). Conversely,
chromosomal translocations (14q+) affecting the IgH locus on 14q32
lead to breakage of the C-J linkage and the two sets of sequences
appear on distinct restriction fragments. (FIG. 11B) Table 1 shows
the application of this analysis to a panel of MM cell lines and
biopsies. The results show that at least 65% of cases show breakage
of the C-J linkage within Ig J or switch regions. The restiction
fragments containing either C or J sequences (R in Table 1) can be
cloned as shown for the SK-MM-1 and U-266 cell lines and the genes
flanking the chromosomal breakpoints can be used as probes to
screen additional MM cases for similar rearrangements, whereas the
sequence of the genes can be used to understand the consequences of
these genetic lesions in multiple myeloma. Cloning of the
chromosomal breakpoints and corresponding genes is currently
ongoing for all of the MM cases shown in Table 1.
[0113] The method of analysis of 14q+chromosomal translocations and
identification of the genes altered in multiple myeloma of this
invention will allow 1) the determination of chromosomal sequences
involved in 14q+translocations, the most important cytogenetic
lesion associated with MM pathogenesis elucidation; 2) elucidation
of specific gene lesions for MM; 3) a diagnostic method based on
gene/DNA lesion and 4) a therapeutic approach aimed at
counteracting the action of abnormal gene products. TABLE-US-00001
TABLE 1 Summary of JH-C breakage analysis in MM cell lines and
biopsies (cases). Rearrangement (R) involving physiologic Ig
recombinations, i.e. retaining JH-C linkage are marked as R*;
rearrangements lacking JH-C linkage, and therefore suggesting a
14q+ chromosomal breakpoint, are marked as R. The latter represents
candidates for cloning an further analysis. possible Cell Line/Case
sIg JH C.mu. C.alpha. S.gamma.3' breakpoint locus RPMI-8226 .lamda.
D/D D/D G R/G S.gamma. U-266 E.lamda. R/D D/D R/R/G G S.alpha. EJM
G.lamda. R/R* D/D G R*/G JH.about.S.mu. XG-1 A.kappa. R*/D D/D R* G
ND XG-2 G.lamda. R*/D D/D G R*/G ND XG-4 G.kappa. R*/R D/D G R*/G
JH.about.S.mu. XG-5 .lamda. R/D D/D G G JH.about.S.mu. XG-6
G.lamda. R*/R D/D G R*/G JH.about.S.mu. XG-7 A.kappa. R/D D/D R/G
R/D S.gamma. XG-10 G R*/R D/D G R*/G JH.about.S.mu. SK-MM-1 .kappa.
R*/R R/D G R*/G JH.about.S.mu. CASE125 R* G G R* ND CASE33 R/R* G G
R/R* S.gamma. CASE34 R* G R* G ND CASE93 R* G R* G ND CASE91 R* R*
R G S.alpha. CASE128 R* G R* G ND R*, comigrated bands with JH; R,
target bands to isolate; ND, not determined
[0114] Possible Breakage in Switch Regions: TABLE-US-00002 Cell
Lines 4/11 (36%) Cases 2/6 (33%) Total 6/17 (35%)
[0115] Possible Breakage in JH.about.Switch Regions: TABLE-US-00003
Cell Lines 9/11 (82%) Cases 2/6 (33%) Total 11/17 (65%)
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Sequence CWU 1
1
18 1 108 PRT Homo sapiens 1 Lys Leu Arg Gln Trp Leu Ile Asp Gln Ile
Asp Ser Gly Lys Tyr Pro 1 5 10 15 Gly Leu Val Trp Glu Asn Glu Glu
Lys Ser Ile Phe Arg Ile Pro Trp 20 25 30 Lys His Ala Gly Lys Gln
Asp Tyr Asn Arg Glu Glu Asp Ala Ala Leu 35 40 45 Phe Lys Ala Trp
Ala Leu Phe Lys Gly Lys Phe Arg Glu Gly Ile Asp 50 55 60 Lys Pro
Asp Pro Pro Thr Trp Lys Thr Arg Leu Arg Cys Ala Leu Asn 65 70 75 80
Lys Ser Asn Asp Phe Glu Glu Leu Val Glu Arg Ser Gln Leu Asp Ile 85
90 95 Ser Asp Pro Tyr Lys Val Tyr Arg Ile Val Pro Glu 100 105 2 108
PRT Mus sp. 2 Lys Leu Arg Gln Trp Leu Ile Asp Gln Ile Asp Ser Gly
Lys Tyr Pro 1 5 10 15 Gly Leu Val Trp Glu Asn Glu Glu Lys Ser Val
Phe Arg Ile Pro Trp 20 25 30 Lys His Ala Gly Lys Gln Asp Tyr Asn
Arg Glu Glu Asp Ala Ala Leu 35 40 45 Phe Lys Ala Trp Ala Leu Phe
Lys Gly Lys Phe Arg Glu Gly Ile Asp 50 55 60 Lys Pro Asp Pro Pro
Thr Trp Lys Thr Arg Leu Arg Cys Ala Leu Asn 65 70 75 80 Lys Ser Asn
Asp Phe Glu Glu Leu Val Glu Arg Ser Gln Leu Asp Ile 85 90 95 Ser
Asp Pro Tyr Lys Val Tyr Arg Ile Val Pro Glu 100 105 3 108 PRT Homo
sapiens 3 Arg Met Arg Pro Trp Leu Glu Met Gln Ile Asn Ser Asn Gln
Ile Pro 1 5 10 15 Gly Leu Ile Trp Ile Asn Lys Glu Glu Met Ile Phe
Gln Ile Pro Trp 20 25 30 Lys His Ala Ala Lys His Gly Trp Asp Ile
Asn Lys Asp Ala Cys Leu 35 40 45 Phe Arg Ser Trp Ala Ile His Thr
Gly Arg Tyr Lys Ala Gly Glu Lys 50 55 60 Glu Pro Asp Pro Lys Thr
Trp Lys Ala Asn Phe Arg Cys Ala Met Asn 65 70 75 80 Ser Leu Pro Asp
Ile Glu Glu Val Lys Asp Gln Ser Arg Asn Lys Gly 85 90 95 Ser Ser
Ala Val Arg Val Tyr Arg Met Leu Pro Pro 100 105 4 108 PRT Homo
sapiens 4 Arg Met Arg Pro Trp Leu Glu Glu Gln Ile Asn Ser Asn Thr
Ile Pro 1 5 10 15 Gly Leu Lys Trp Leu Asn Lys Glu Lys Lys Ile Phe
Gln Ile Pro Trp 20 25 30 Met His Ala Ala Arg His Gly Trp Asp Val
Glu Lys Asp Ala Pro Leu 35 40 45 Phe Arg Asn Trp Ala Ile His Thr
Gly Lys His Gln Pro Gly Val Asp 50 55 60 Lys Pro Asp Pro Lys Thr
Trp Lys Ala Asn Phe Arg Cys Ala Met Asn 65 70 75 80 Ser Leu Pro Asp
Ile Glu Glu Val Lys Asp Lys Ser Ile Lys Lys Gly 85 90 95 Asn Asn
Ala Phe Arg Val Tyr Arg Met Leu Pro Leu 100 105 5 107 PRT Homo
sapiens 5 Arg Leu Arg Gln Trp Leu Ile Glu Gln Ile Asp Ser Ser Met
Tyr Pro 1 5 10 15 Gly Leu Ile Trp Glu Asn Glu Glu Lys Ser Met Phe
Arg Ile Pro Trp 20 25 30 Lys His Ala Gly Lys Gln Asp Tyr Asn Gln
Glu Val Asp Ala Ser Ile 35 40 45 Phe Lys Ala Trp Ala Val Phe Lys
Gly Lys Phe Lys Glu Gly Asp Lys 50 55 60 Ala Glu Pro Ala Thr Trp
Lys Thr Arg Leu Arg Cys Ala Leu Asn Lys 65 70 75 80 Ser Pro Asp Phe
Glu Glu Val Thr Asp Arg Ser Gln Leu Asp Ile Ser 85 90 95 Glu Pro
Tyr Lys Val Tyr Arg Ile Val Pro Glu 100 105 6 107 PRT Homo sapiens
6 Lys Leu Arg Asn Trp Val Val Glu Gln Val Glu Ser Gly Gln Phe Pro 1
5 10 15 Gly Val Cys Trp Asp Asp Thr Ala Lys Thr Met Phe Arg Ile Pro
Trp 20 25 30 Lys His Ala Gly Lys Gln Asp Phe Arg Glu Asp Gln Asp
Ala Ala Phe 35 40 45 Phe Lys Ala Trp Ala Ile Phe Lys Gly Lys Tyr
Lys Glu Gly Asp Thr 50 55 60 Gly Gly Pro Ala Val Trp Lys Thr Arg
Leu Arg Cys Ala Leu Asn Lys 65 70 75 80 Ser Ser Glu Phe Lys Glu Val
Pro Glu Arg Gly Arg Met Asp Val Ala 85 90 95 Glu Pro Tyr Lys Val
Tyr Gln Leu Leu Pro Pro 100 105 7 106 PRT Homo sapiens 7 Arg Ile
Leu Pro Trp Leu Val Ser Gln Leu Asp Leu Gly Gln Leu Glu 1 5 10 15
Gly Val Ala Trp Val Asn Lys Ser Arg Thr Arg Phe Arg Ile Pro Trp 20
25 30 Lys His Gly Leu Arg Gln Asp Ala Gln Gln Glu Asp Phe Gly Ile
Phe 35 40 45 Gln Ala Trp Ala Glu Ala Thr Gly Ala Tyr Val Pro Gly
Arg Asp Lys 50 55 60 Pro Asp Leu Pro Thr Trp Lys Arg Asn Phe Arg
Ser Ala Leu Asn Arg 65 70 75 80 Lys Glu Gly Leu Arg Leu Ala Glu Asp
Arg Ser Lys Asp Pro His Asp 85 90 95 Pro His Lys Ile Tyr Glu Phe
Val Asn Ser 100 105 8 95 PRT Homo sapiens 8 Lys Arg Leu Cys Gln Ser
Thr Ile Tyr Trp Asp Gly Pro Leu Ala Leu 1 5 10 15 Cys Asn Asp Arg
Pro Asn Lys Leu Glu Arg Asp Gln Thr Cys Lys Leu 20 25 30 Phe Asp
Thr Gln Gln Phe Leu Ser Glu Leu Gln Ala Phe Ala His His 35 40 45
Gly Arg Ser Leu Pro Arg Phe Gln Val Thr Leu Cys Phe Gly Glu Glu 50
55 60 Phe Pro Asp Pro Gln Arg Gln Arg Lys Leu Ile Thr Ala His Val
Glu 65 70 75 80 Pro Leu Leu Ala Arg Gln Leu Tyr Tyr Phe Ala Gln Gln
Asn Ser 85 90 95 9 95 PRT Mus sp. 9 Lys Arg Leu Cys Gln Ser Arg Ile
Tyr Trp Asp Gly Pro Leu Ala Leu 1 5 10 15 Cys Ser Asp Arg Pro Asn
Lys Leu Glu Arg Asp Gln Thr Cys Lys Leu 20 25 30 Phe Asp Thr Gln
Gln Phe Leu Ser Glu Leu Gln Val Phe Ala His His 35 40 45 Gly Arg
Pro Ala Pro Arg Phe Gln Val Thr Leu Cys Phe Gly Glu Glu 50 55 60
Phe Pro Asp Pro Gln Arg Gln Arg Lys Leu Ile Thr Ala His Val Glu 65
70 75 80 Pro Leu Leu Ala Arg Gln Leu Tyr Tyr Phe Ala Gln Gln Asn
Thr 85 90 95 10 96 PRT Homo sapiens 10 Lys Arg Leu Cys Gln Gly Arg
Val Phe Cys Ser Gly Asn Ala Val Val 1 5 10 15 Cys Lys Gly Arg Pro
Asn Lys Leu Glu Arg Asp Glu Val Val Gln Val 20 25 30 Phe Asp Thr
Ser Gln Phe Phe Arg Glu Leu Gln Gln Phe Tyr Asn Ser 35 40 45 Gln
Gly Arg Leu Pro Asp Gly Arg Val Val Leu Cys Phe Gly Glu Glu 50 55
60 Phe Pro Asp Met Ala Pro Leu Arg Ser Lys Leu Ile Leu Val Gln Ile
65 70 75 80 Glu Gln Leu Tyr Val Arg Gln Leu Ala Glu Glu Ala Gly Lys
Ser Cys 85 90 95 11 96 PRT Homo sapiens 11 Gln Arg Leu Cys Pro Ile
Pro Ile Ser Trp Asn Ala Pro Gln Ala Pro 1 5 10 15 Pro Gly Pro Gly
Pro His Leu Leu Pro Ser Asn Glu Cys Val Glu Leu 20 25 30 Phe Arg
Thr Ala Tyr Phe Cys Arg Asp Leu Val Arg Tyr Phe Gln Gly 35 40 45
Leu Gly Pro Pro Pro Lys Phe Gln Val Thr Leu Asn Phe Trp Glu Glu 50
55 60 Ser His Gly Ser Ser His Thr Pro Gln Asn Leu Ile Thr Val Lys
Met 65 70 75 80 Glu Gln Ala Phe Ala Arg Tyr Leu Leu Glu Gln Thr Pro
Glu Gln Gln 85 90 95 12 100 PRT Homo sapiens 12 Gln Arg Leu Gly His
Cys His Thr Tyr Trp Ala Val Ser Glu Glu Leu 1 5 10 15 Leu Pro Asn
Ser Gly His Gly Pro Asp Gly Glu Val Pro Lys Asp Lys 20 25 30 Glu
Gly Gly Val Phe Asp Leu Gly Pro Phe Ile Val Asp Leu Ile Thr 35 40
45 Phe Thr Glu Gly Ser Gly Arg Ser Pro Arg Tyr Ala Leu Trp Phe Cys
50 55 60 Val Gly Glu Ser Trp Pro Gln Asp Gln Pro Trp Thr Lys Arg
Leu Val 65 70 75 80 Met Val Lys Val Val Pro Thr Cys Leu Arg Ala Leu
Val Glu Met Ala 85 90 95 Arg Val Gly Gly 100 13 5176 DNA Homo
sapiens CDS (217)..(1569) 13 gcctgaccaa catggtaaaa ccccatctct
gctaaaacta caaaaaatta gctggatgtg 60 gtggcaggga acctgtcatc
ccagctagtt gggagactga ggcaggagaa tcgctcgatc 120 ttgggaccca
ccgctgccct cagctccgag tccagggcga gtgcagagca cagcgggcgg 180
aggaccccgg gcgcgggcgc ggacggcacg cggggc atg aac ctg gag ggc ggc 234
Met Asn Leu Glu Gly Gly 1 5 ggc cga ggc gga gag ttc ggc atg agc gcg
gtg agc tgc ggc aac ggg 282 Gly Arg Gly Gly Glu Phe Gly Met Ser Ala
Val Ser Cys Gly Asn Gly 10 15 20 aag ctc cgc cag tgg ctg atc gac
cag atc gac agc ggc aag tac ccc 330 Lys Leu Arg Gln Trp Leu Ile Asp
Gln Ile Asp Ser Gly Lys Tyr Pro 25 30 35 ggg ctg gtg tgg gag aac
gag gag aag agc atc ttc cgc atc ccc tgg 378 Gly Leu Val Trp Glu Asn
Glu Glu Lys Ser Ile Phe Arg Ile Pro Trp 40 45 50 aag cac gcg ggc
aag cag gac tac aac cgc gag gag gac gcc gcg ctc 426 Lys His Ala Gly
Lys Gln Asp Tyr Asn Arg Glu Glu Asp Ala Ala Leu 55 60 65 70 ttc aag
gct tgg gca ctg ttt aaa gga aag ttc cga gaa ggc atc gac 474 Phe Lys
Ala Trp Ala Leu Phe Lys Gly Lys Phe Arg Glu Gly Ile Asp 75 80 85
aag ccg gac cct ccc acc tgg aag acg cgc ctg cgg tgc gct ttg aac 522
Lys Pro Asp Pro Pro Thr Trp Lys Thr Arg Leu Arg Cys Ala Leu Asn 90
95 100 aag agc aat gac ttt gag gaa ctg gtt gag cgg agc cag ctg gac
atc 570 Lys Ser Asn Asp Phe Glu Glu Leu Val Glu Arg Ser Gln Leu Asp
Ile 105 110 115 tca gac ccg tac aaa gtg tac agg att gtt cct gag gga
gcc aaa aaa 618 Ser Asp Pro Tyr Lys Val Tyr Arg Ile Val Pro Glu Gly
Ala Lys Lys 120 125 130 gga gcc aag cag ctc acc ctg gag gac ccg cag
atg tcc atg agc cac 666 Gly Ala Lys Gln Leu Thr Leu Glu Asp Pro Gln
Met Ser Met Ser His 135 140 145 150 ccc tac acc atg aca acg cct tac
cct tcg ctc cca gcc cag cag gtt 714 Pro Tyr Thr Met Thr Thr Pro Tyr
Pro Ser Leu Pro Ala Gln Gln Val 155 160 165 cac aac tac atg atg cca
ccc ctc gac cga agc tgg agg gac tac gtc 762 His Asn Tyr Met Met Pro
Pro Leu Asp Arg Ser Trp Arg Asp Tyr Val 170 175 180 ccg gat cag cca
cac ccg gaa atc ccg tac caa tgt ccc atg acg ttt 810 Pro Asp Gln Pro
His Pro Glu Ile Pro Tyr Gln Cys Pro Met Thr Phe 185 190 195 gga ccc
cgc ggc cac cac tgg caa ggc cca gct tgt gaa aat ggt tgc 858 Gly Pro
Arg Gly His His Trp Gln Gly Pro Ala Cys Glu Asn Gly Cys 200 205 210
cag gtg aca gga acc ttt tat gct tgt gcc cca cct gag tcc cag gct 906
Gln Val Thr Gly Thr Phe Tyr Ala Cys Ala Pro Pro Glu Ser Gln Ala 215
220 225 230 ccc gga gtc ccc aca gag cca agc ata agg tct gcc gaa gcc
ttg gcg 954 Pro Gly Val Pro Thr Glu Pro Ser Ile Arg Ser Ala Glu Ala
Leu Ala 235 240 245 ttc tca gac tgc cgg ctg cac atc tgc ctg tac tac
cgg gaa atc ctc 1002 Phe Ser Asp Cys Arg Leu His Ile Cys Leu Tyr
Tyr Arg Glu Ile Leu 250 255 260 gtg aag gag ctg acc acg tcc agc ccc
gag ggc tgc cgg atc tcc cat 1050 Val Lys Glu Leu Thr Thr Ser Ser
Pro Glu Gly Cys Arg Ile Ser His 265 270 275 gga cat acg tat gac gcc
agc aac ctg gac cag gtc ctg ttc ccc tac 1098 Gly His Thr Tyr Asp
Ala Ser Asn Leu Asp Gln Val Leu Phe Pro Tyr 280 285 290 cca gag gac
aat ggc cac agg aaa aac att gag aac ctg ctg agc cac 1146 Pro Glu
Asp Asn Gly His Arg Lys Asn Ile Glu Asn Leu Leu Ser His 295 300 305
310 ctg gag agg ggc gtg gtc ctc tgg atg gcc ccc gac ggg ctc tat gcg
1194 Leu Glu Arg Gly Val Val Leu Trp Met Ala Pro Asp Gly Leu Tyr
Ala 315 320 325 aaa aga ctg tgc cag agc acg atc tac tgg gac ggg ccc
ctg gcg ctg 1242 Lys Arg Leu Cys Gln Ser Thr Ile Tyr Trp Asp Gly
Pro Leu Ala Leu 330 335 340 tgc aac gac cgg ccc aac aaa ctg gag aga
gac cag acc tgc aag ctc 1290 Cys Asn Asp Arg Pro Asn Lys Leu Glu
Arg Asp Gln Thr Cys Lys Leu 345 350 355 ttt gac aca cag cag ttc ttg
tca gag ctg caa gcg ttt gct cac cac 1338 Phe Asp Thr Gln Gln Phe
Leu Ser Glu Leu Gln Ala Phe Ala His His 360 365 370 ggc cgc tcc ctg
cca aga ttc cag gtg act cta tgc ttt gga gag gag 1386 Gly Arg Ser
Leu Pro Arg Phe Gln Val Thr Leu Cys Phe Gly Glu Glu 375 380 385 390
ttt cca gac cct cag agg caa aga aag ctc atc aca gct cac gta gaa
1434 Phe Pro Asp Pro Gln Arg Gln Arg Lys Leu Ile Thr Ala His Val
Glu 395 400 405 cct ctg cta gcc aga caa cta tat tat ttt gct caa caa
aac agt gga 1482 Pro Leu Leu Ala Arg Gln Leu Tyr Tyr Phe Ala Gln
Gln Asn Ser Gly 410 415 420 cat ttc ctg agg ggc tac gat tta cca gaa
cac atc agc aat cca gaa 1530 His Phe Leu Arg Gly Tyr Asp Leu Pro
Glu His Ile Ser Asn Pro Glu 425 430 435 gat tac cac aga tct atc cgc
cat tcc tct att caa gaa tgaaaaatgt 1579 Asp Tyr His Arg Ser Ile Arg
His Ser Ser Ile Gln Glu 440 445 450 caagatgagt ggttttcttt
ttcctttttt tttttttttt ttttgatacg gagatacggg 1639 gtcttgctct
gtctcccagg ctggagtgca gtgacacaat ctcagctcac tgtgacctcc 1699
gcctcctggg ttcaagagac tctcctgcct cagcctccct ggtagctggg attacaggtg
1759 tgagccactg cacccaccca agacaagtga ttttcattgt aaatatttga
ctttagtgaa 1819 agcgtccaat tgactgccct cttactgttt tgaggaactc
agaagtggag atttcagttc 1879 agcggttgag gagaattgcg gcgagacaag
catggaaaat cagtgacatc tgattggcag 1939 atgagcttat ttcaaaagga
agggtggctt tgcattttct tgtgttctgt agactgccat 1999 cattgatgat
cactgtgaaa attgaccaag tgatgtgttt acatttactg aaatgcgctc 2059
tttaatttgt tgtagattag gtcttgctgg aagacagaga aaacttgcct ttcagtattg
2119 acactgacta gagtgatgac tgcttgtagg tatgtctgtg ccatttctca
gggaagtaag 2179 atgtaaattg aagaagcctc acacgtaaaa gaaatgtatt
aatgtatgta ggagctgcag 2239 ttcttgtgga agacacttgc tgagtgaagg
aaatgaatct ttgactgaag ccgtgcctgt 2299 agccttgggg aggcccatcc
cccacctgcc agcggtttcc tggtgtgggt ccctctgccc 2359 caccctcctt
cccattggct ttctctcctt ggcctttcct ggaagccagt tagtaaactt 2419
cctattttct tgagtcaaaa aacatgagcg ctactcttgg atgggacatt tttgtctgtc
2479 ctacaatcta gtaatgtcta agtaatggtt aagttttctt gtttctgcat
ctttttgacc 2539 ctcattcttt agagatgcta aaattcttcg cataaagaag
aagaaattaa ggaacataaa 2599 tcttaatact tgaactgttg cccttctgtc
caagtactta actatctgtt cccttcctct 2659 gtgccacgct cctctgtttg
tttggctgtc cagcgatcag ccatggcgac actaaaggag 2719 gaggagccgg
ggactcccag gctggagagc actgccagga cccaccactg gaagcaggat 2779
ggagctgact acggaactgc acactcagtg ggctgtttct gcttatttca tctgttctat
2839 gcttcctcgt gccaattata gtttgacagg gccttaaaat tacttggctt
tttccaaatg 2899 cttctattta tagaaatccc aaagacctcc acttgcttaa
gtatacctat cacttacatt 2959 tttgtggttt tgagaaagta cagcagtaga
ctggggcgtc acctccaggc cgtttctcat 3019 actacaggat atttactatt
actcccagga ttcagcagaa gattgcgtta gctctcaaat 3079 gtgtgttcct
gcttttctaa tggatatttt aaattcattc aacaagcacc tagtaagtgc 3139
ctgctgtatc cctacattac acagttcagc ctttatcaag cttagtgagc agtgagcact
3199 gaaacattat tttttaatgt ttaaaaagtt tctaatatta aagtcagaat
attaatacaa 3259 ttaatattaa tattaactac agaaaagaca aacagtagag
aacagcaaaa aaataaaaag 3319 gatctccttt tttcccagcc caaattctcc
tctctaaaag tgtccacaag aaggggtgtt 3379 tattcttcca acacatttca
cttttctgta aatatacata aacttaaaaa gaaaacctca 3439 tggagtcatc
ttgcacacac ttttcatgca gtgctctttg tagctaaaca gtgaagattt 3499
acctcgttct gctcagaggc cttgctgtgg agctccactg ccatgtaccc agtagggttt
3559 gacatttcat tagccatgca acatggatat gtattgggca gcagactgtg
tttcgtgaac 3619 tgcagtgatg tatacatctt atagatgcaa agtattttgg
ggtatattat cctaagggaa 3679 gataaagatg atattaagaa ctgctgtttc
acggggccct tacctgtgac cctctttgct 3739 gaagaatatt taaccccaca
cagcacttca aagaagctgt cttggaagtc tgtctcagga 3799 gcaccctgtc
ttcttaattc tccaagcgga tgctccattt caattgcttt gtgacttctt 3859
cttctttgtt tttttaaata ttatgctgct ttaacagtgg agctgaattt tctggaaaat
3919 gcttcttggc tggggccact acctcctttc ctatctttac atctatgtgt
atgttgactt 3979 tttaaaattc tgagtgatcc
agggtatgac ctagggaatg aactagctat ggaaataact 4039 cagggttagg
aatcctagca cttgtctcag gactctgaaa aggaacggct tcctcattcc 4099
ttgtcttgat aaagtggaat tggcaaacta gaatttagtt tgtactcagt ggacagtgct
4159 gttgaagatt tgaggacttg ttaaagagca ctgggtcata tggaaaaaat
gtatgtgtct 4219 ccccaggtgc attttcttgg tttatgtctt gttcttgaga
ttttgtatat ttaggaaaac 4279 ctcaagcagt aattaatatc tcctggaaca
ctatagagaa ccaagtgacc gactcattta 4339 caactgaaac ctaggaagcc
cctgagtcct gagcgaaaac aggagagtta gtcgccctac 4399 agaaaaccca
gctagactat tgggtatgaa ctaaaaagag actgtgccat ggtgagaaaa 4459
atgtaaaatc ctacagtgga atgagcagcc cttacagtgt tgttaccacc aagggcaggt
4519 aggtattagt gtttgaaaaa gctggtcttt gagcgagggc ataaatacag
ctagccccag 4579 gggtggaaca actgtgggag tcttgggtac tcgcacctct
tggctttgtt gatgctccgc 4639 caggaaggcc acttgtgtgt gcgtgtcagt
tactttttta gtaacaattc agatccagtg 4699 taaacttccg ttcattgctc
tccagtcaca tgcccccact tccccacagg tgaaagtttt 4759 tctgaagtgt
tgggattggt taaggtcttt atttgtatta cgtatctccc caagtcctct 4819
gtggccagct gcatctgtct gaatggtgcg tgaaggctct cagaccttac acaccatttt
4879 gtaagttatg ttttacatgc cccgtttttg agactgatct cgatgcaggt
ggatctcctt 4939 gagatcctga tagcctgtta caggaatgaa gtaaaggtca
gttttttttg tattgatttt 4999 cacagctttg aggaacatgc ataagaaatg
tagctgaagt agaggggacg tgagagaagg 5059 gccaggccgg caggccaacc
ctcctccaat ggaaattccc gtgttgcttc aaactgagac 5119 agatgggact
taacaggcaa tggggtccac ttccccctct tcagcatccc ccgtacc 5176 14 451 PRT
Homo sapiens 14 Met Asn Leu Glu Gly Gly Gly Arg Gly Gly Glu Phe Gly
Met Ser Ala 1 5 10 15 Val Ser Cys Gly Asn Gly Lys Leu Arg Gln Trp
Leu Ile Asp Gln Ile 20 25 30 Asp Ser Gly Lys Tyr Pro Gly Leu Val
Trp Glu Asn Glu Glu Lys Ser 35 40 45 Ile Phe Arg Ile Pro Trp Lys
His Ala Gly Lys Gln Asp Tyr Asn Arg 50 55 60 Glu Glu Asp Ala Ala
Leu Phe Lys Ala Trp Ala Leu Phe Lys Gly Lys 65 70 75 80 Phe Arg Glu
Gly Ile Asp Lys Pro Asp Pro Pro Thr Trp Lys Thr Arg 85 90 95 Leu
Arg Cys Ala Leu Asn Lys Ser Asn Asp Phe Glu Glu Leu Val Glu 100 105
110 Arg Ser Gln Leu Asp Ile Ser Asp Pro Tyr Lys Val Tyr Arg Ile Val
115 120 125 Pro Glu Gly Ala Lys Lys Gly Ala Lys Gln Leu Thr Leu Glu
Asp Pro 130 135 140 Gln Met Ser Met Ser His Pro Tyr Thr Met Thr Thr
Pro Tyr Pro Ser 145 150 155 160 Leu Pro Ala Gln Gln Val His Asn Tyr
Met Met Pro Pro Leu Asp Arg 165 170 175 Ser Trp Arg Asp Tyr Val Pro
Asp Gln Pro His Pro Glu Ile Pro Tyr 180 185 190 Gln Cys Pro Met Thr
Phe Gly Pro Arg Gly His His Trp Gln Gly Pro 195 200 205 Ala Cys Glu
Asn Gly Cys Gln Val Thr Gly Thr Phe Tyr Ala Cys Ala 210 215 220 Pro
Pro Glu Ser Gln Ala Pro Gly Val Pro Thr Glu Pro Ser Ile Arg 225 230
235 240 Ser Ala Glu Ala Leu Ala Phe Ser Asp Cys Arg Leu His Ile Cys
Leu 245 250 255 Tyr Tyr Arg Glu Ile Leu Val Lys Glu Leu Thr Thr Ser
Ser Pro Glu 260 265 270 Gly Cys Arg Ile Ser His Gly His Thr Tyr Asp
Ala Ser Asn Leu Asp 275 280 285 Gln Val Leu Phe Pro Tyr Pro Glu Asp
Asn Gly His Arg Lys Asn Ile 290 295 300 Glu Asn Leu Leu Ser His Leu
Glu Arg Gly Val Val Leu Trp Met Ala 305 310 315 320 Pro Asp Gly Leu
Tyr Ala Lys Arg Leu Cys Gln Ser Thr Ile Tyr Trp 325 330 335 Asp Gly
Pro Leu Ala Leu Cys Asn Asp Arg Pro Asn Lys Leu Glu Arg 340 345 350
Asp Gln Thr Cys Lys Leu Phe Asp Thr Gln Gln Phe Leu Ser Glu Leu 355
360 365 Gln Ala Phe Ala His His Gly Arg Ser Leu Pro Arg Phe Gln Val
Thr 370 375 380 Leu Cys Phe Gly Glu Glu Phe Pro Asp Pro Gln Arg Gln
Arg Lys Leu 385 390 395 400 Ile Thr Ala His Val Glu Pro Leu Leu Ala
Arg Gln Leu Tyr Tyr Phe 405 410 415 Ala Gln Gln Asn Ser Gly His Phe
Leu Arg Gly Tyr Asp Leu Pro Glu 420 425 430 His Ile Ser Asn Pro Glu
Asp Tyr His Arg Ser Ile Arg His Ser Ser 435 440 445 Ile Gln Glu 450
15 154 DNA Homo sapiens 15 ttttctctct acagtcacct ccctgtttac
caaagataat cacaataagt ccagtttact 60 tacaaaacaa gtttagttat
tagaggaaac taaaacttca ggattcagtc cagataattt 120 ttaaaaactc
taaaacaatg gacagggcta gaat 154 16 75 DNA Homo sapiens 16 tgggctcggc
ctggtggggc agccacagcg ggacgcagta gtgaaagtcc agtttactta 60
caaaacaagt ttagt 75 17 77 DNA Homo sapiens 17 tattagagga aactaaaact
tcaggattca gcagggcatg aggaggcagc tcctcaccct 60 ccctttctct tttgtac
77 18 122 DNA Homo sapiens 18 tgggctcggc cttggtgggg cagccacagc
gggacgcaag tagtgagggc actcagaacg 60 ccactcagcc ccgacaggca
gggcacgagg aggcagctcc tcaccctccc tttctctttt 120 gt 122
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