U.S. patent application number 09/749273 was filed with the patent office on 2004-09-16 for novel chromosome 21 gene marker, compositions and methods using same.
Invention is credited to Korenberg, Julie R., Yamakawa, Kazuhiro.
Application Number | 20040180335 09/749273 |
Document ID | / |
Family ID | 23321594 |
Filed Date | 2004-09-16 |
United States Patent
Application |
20040180335 |
Kind Code |
A1 |
Korenberg, Julie R. ; et
al. |
September 16, 2004 |
Novel chromosome 21 gene marker, compositions and methods using
same
Abstract
The present invention provides isolated nucleic acids encoding
human EHOC-1 protein and isolated receptor proteins encoded
thereby. Further provided are vectors containing invention nucleic
acids, probes that hybridize thereto, host cells transformed
therewith, antisense oligonucleotides thereto and compositions
containing, antibodies that specifically bind to invention
polypeptides and compositions containing, as well as transgenic
non-human mammals that express the invention protein.
Inventors: |
Korenberg, Julie R.; (Los
Angeles, CA) ; Yamakawa, Kazuhiro; (Los Angeles,
CA) |
Correspondence
Address: |
PENNIE AND EDMONDS
1155 AVENUE OF THE AMERICAS
NEW YORK
NY
100362711
|
Family ID: |
23321594 |
Appl. No.: |
09/749273 |
Filed: |
December 26, 2000 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09749273 |
Dec 26, 2000 |
|
|
|
09048887 |
Mar 26, 1998 |
|
|
|
09048887 |
Mar 26, 1998 |
|
|
|
08337690 |
Nov 9, 1994 |
|
|
|
Current U.S.
Class: |
435/6.11 ;
435/183; 435/320.1; 435/325; 435/6.13; 435/69.1; 506/9;
536/23.2 |
Current CPC
Class: |
C07K 14/47 20130101;
A01K 2217/05 20130101; A61K 48/00 20130101 |
Class at
Publication: |
435/006 ;
435/183; 435/069.1; 435/325; 435/320.1; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/00; C12P 021/02; C12N 005/06 |
Goverment Interests
[0001] This invention was made in part with Government support
under Grant No. HD17449-11, from the National Institutes of Child
Health and Human Development. The Government may have certain
rights in this invention.
Claims
What is claimed is:
1. Isolated nucleic acid encoding a human EHOC-1 polypeptide.
2. Isolated nucleic acid according to claim 1, wherein said nucleic
comprises DNA.
3. DNA according to claim 2, wherein said DNA is a cDNA.
4. DNA according to claim 2, wherein said DNA encodes the amino
acid sequence set forth in SEQ ID NO: 2.
5. DNA according to claim 2, wherein said DNA hybridizes under high
stringency coditions to substantially the entire coding sequence
(nucleotides 157-3726) set forth in SEQ ID NO: 2.
6. DNA according to claim 2, wherein said DNA has substantially the
same nucleotide sequence as the nucleotide sequence set forth in
SEQ ID NO: 1.
7. A vector comprising DNA according to claim 2.
8. A host cell containing a vector according to claim 7, wherein
said cell is a procaryotic cell or a eucaryotic cell.
9. A host cell according to claim 8, wherein said cell expresses a
functional EHOC-1 protein.
10. A nucleic acid probe comprising at least 15 nucleotides capable
of specifically hybridizing with a sequence of nucleic acids of the
nucleotide sequence set forth in SEQ ID NO: 1.
11. A nucleic acid probe according to claim 10, wherein said probe
is labeled with a detectable marker.
12. A kit for detecting mutations and aneuploidies in chromosome 21
at locus q22.3 comprising a plurality of probes, wherein each probe
comprises a nucleic acid sequence having at least 15 bp of
contiguous nucleotides capable of specifically hybridizing with a
sequence of nucleic acids of the nucleotide sequence set forth in
SEQ ID NO: 1, and wherein each individual probe corresponds to a
specific locus on chromosome 21q22.3
13. Isolated mRNA complementary to DNA according to claim 2.
14. An oligonucleotide composition comprising chemical analogues of
the nucleic acid of claim 2 operatively linked to a promoter of RNA
transcription.
15. An antisense oligonucleotide capable of specifically binding to
and modulating the translation of mRNA according to claim 13.
16. Isolated EHOC-1 polypeptide and functional equivalents
thereof.
17. Isolated EHOC-1 polypeptide according to claim 16, wherein said
polypeptide has substantially the same amino acid sequence as that
set forth in SEQ ID NO: 2.
18. Isolated EHOC-1 polypeptide according to claim 16, wherein said
polypeptide has the same amino acid sequence as that set forth in
SEQ ID NO: 2.
19. Isolated EHOC-1 polypeptide according to claim 16, wherein said
polypeptide is encoded by a nucleotide sequence that is
substantially the same nucleotide sequence as that set forth in SEQ
ID NO: 1.
20. Isolated EHOC-1 polypeptide according to claim 16, wherein said
polypeptide is encoded by the nucleotide sequence set forth in SEQ
ID NO: 1.
21. An EHOC-1 polypeptide expressed recombinantly in a host
cell.
22. An EHOC-1 polypeptide according to claim 21, wherein said
polypeptide is encoded by a nucleotide sequence that is
substantially the same as the nucleotide sequence set forth in SEQ
ID NO: 1.
23. An EHOC-1 polypeptide according to claim 21, wherein said
polypeptide is encoded by the nucleotide sequence set forth in SEQ
ID NO: 1.
24. An antibody that specifically binds to a determinant on a human
EHOC-1 protein or active fragment thereof.
25. An antibody according to claim 24, wherein said antibody is a
monoclonal antibody.
26. An antibody according to claim 24, wherein said antibody is a
polyclonal antibody.
27. A composition comprising an amount of the antisense
oligonucleotide according to claim 13 effective to modulate
expression of a human EHOC-1 polypeptide and an acceptable
hydrophobic carrier capable of passing through a cell membrane.
28. A composition according to claim 27, wherein the
oligonucleotide is coupled to a substance which inactivates
mRNA.
29. A composition according to claim 28, wherein said substance is
a ribozyme.
30. A composition comprising an amount of an antibody according to
claim 24 effective to block binding of naturally occurring ligands
to the human EHOC-1 receptor and an acceptable carrier.
31. A transgenic nonhuman mammal expressing DNA encoding a human
EHOC-1 polypeptide.
32. A transgenic nonhuman mammal according to claim 31, wherein
said DNA encoding said polypeptide has been mutated as to be
incapable of normal polypeptide activity, and wherein the
polypeptide so expressed is not native EHOC-1 polypeptide.
33. A transgenic nonhuman mammal, the genome of which comprising
antisense DNA complementary to DNA encoding a human EHOC-1
polypeptide, wherein said antisense DNA s transcribed into
antisense mRNA complementary to mRNA encoding a human EHOC-1
polypeptide.
34. A transgenic nonhuman mammal according to claim 31, wherein
said DNA -s operatively linked to an inducible promoter.
35. A transgenic nonhuman mammal according to claim 31, wherein
said DNA is operatively linked to tissue specific regulatory
elements.
36. A transgenic nonhuman mammal according to claim 31, wherein the
transgenic nonhuman mammal is a mouse.
37. A method for identifying nucleic acids encoding a human EHOC-1
protein, said method comprising: contacting a sample containing
nucleic acids with a probe according to claim 11, wherein said
contacting is effected under high stringency hybridization
conditions, and identifying compounds which hybridize thereto.
38. A method for identifying compound(s) which bind to a human
EHOC-1 polypeptide, said method comprising contacting cells
according to claim 9 with said compound(s) and identifying
compounds which bind thereto.
39. A method for detecting the presence of a human EHOC-1
polypeptide on a cell surface, said method comprising contacting a
test cell with an antibody according to claim 24, detecting the
presence of an antibody-receptor complex, and therefor detecting
the presence of a human EHOC-1 polypeptide on the cell surface.
40. A method for diagnosing a predisposition to a disorder
associated with the expression of a specific human EHOC-1
polypeptide allele, said method comprising: contacting a sample
containing nucleic acids with a plurality of probes, wherein each
probe comprises a nucleic acid sequence having at least 15 bp of
contiguous nucleotides capable of specifically hybridizing with a
sequence of nucleic acids of the nucleotide sequence set forth in
SEQ ID NO: 1, and wherein each individual probe corresponds to a
specific locus on chromosome 21q22.3
41. A method according to claim 40, wherein said disorder is
selected from progressive myoclonus epilepsy, holoprosencephaly, or
autoimmune polyglandular disease.
42. A method for deterring the onset of symptoms associated faith
particular disorder comprising administering a composition which
modulates expression of gene.
43. A method for introducing changes at human chromosome locus
21q22.3 comprising transforming a sample of cells obtained from a
subject having progressive myoclonus epilepsy with the nucleic acid
according to claim 1 along with a selective marker gene;
maintaining cells in selective media; and isolating viable cells
containing a modified target sequence.
44. A method of supplying wild-type EHOC-1 gene function to a cell
which has a mutation/aneuploidy in the EHOC-1 gene comprising
introducing a wild-type EHOC-1 gene or functional fragment thereof
into said cell such that it is expressed.
45. Single strand DNA primers for amplification diagnosis of
progressive myoclonus epilepsy, wherein said primers comprise a
nucleic acid sequence derived from the nucleic acid sequence set
forth as SEQ TD NO: 1.
46. A method for detecting one or more EHOC-1 alleles in a sample
of nucleic acid comprising determining the presence or absence of
variant nuceotide sequence in a gene contained in any of BAC
clones.
Description
BACKGROUND OF THE INVENTION
[0002] A major endeavor in molecular genetics has been made in
generating maps of the human genome. Human genome mapping consists,
generally, of ordering genomic DNA fragments on their chromosomes
using several methods, such as fluorescent n situ hybridization
(FISH), somatic cell hybrid analysis or random clone
fingerprinting. DNA fragments that correspond to marked polymorphic
sites can be ordered by genetic linkage analysis. Distances between
polymorphic loci are estimated by meiotic recombination
frequencies. High resolution maps based upon the estimated
distances, however, cannot be constructed easily using such methods
because the resolution is low at the molecular level and
recombination frequency is not linearly correlated with physical
distance.
[0003] Thus, various obstacles such as, for example, the difficulty
in obtaining highly informative markers and the paucity of
identified markers that are evenly spaced along the chromosome are
significant weaknesses of the currently available genetic maps.
Most of the mapped markers are restriction fragment length
polymorphisms (RFPLs) assayed by DNA hybridization. Although maps
based on these markers have contributed greatly to the primary
mapping of a number of diseases, they are still insufficient for
many applications such as mapping rare monofactorial diseases,
refining linkage intervals to distances suited for gene
identification, and mapping of loci contributing to complex
traits.
[0004] Genetic linkage mapping is an important technology applied
to the study of human biology and, in particular, for the
delineation of the molecular basis of disease. Indeed, one of the
most commonly used strategies for studying human inherited diseases
is by cloning the responsible gene based on chromosomal location.
Genetic linkage maps, therefore, facilitate the identification and
mapping of genes involved in monogenic diseases, genes involved in
multifactorial disorders, and are useful in carrier detection and
prenatal diagnosis of hereditary disorders. A detailed linkage map
is also a prerequisite for clone-based physical mapping and DNA
sequencing of the entire chromosome.
[0005] Human chromosome 21 is a paradigm for large-scale human
genome mappind efforts. The smallest human chromosome, chromosome
21 has approximately 50 megabases (Mb) of DNA. Less than 1% of the
2000 genes estimated to be on chromosome 21 are known. A high
resolution map of chromosomes of particular interest because of is
apparent role in familial Alzheimer disease (FAD), Down's syndrome
(DS), amyotrophic lateral sclerosis (ALS), and Finnish progressive
myoclonus epilepsy (PME). A gene defect responsible for FAD has
been localized to chromosome 21 on the basis of genetic linkage to
three pericentromeric loci. The gene encoding the precursor of the
Alzheimer-associated amyloid .beta. protein (APP), the principle
component of the senile plaques and cerebrovascular amyloid
deposits of Alzheimer disease (AD), has also been mapped to
chromosome 21.
[0006] The process of developing such a long-range contig map
involves the identification and localization of landmarks in cloned
genetic fragments. When there are enough landmarks for the size of
the cloned fragments, contigs are formed, and the landmarks are
simultaneously ordered. Currently, YACs, or yeast artificial
chromosomes, are utilized for most mapping of the human genome.
YACs permit cloning of fragments of .gtoreq. about 500 Kb. However,
some difficulties have been encountered with the manipulation of
YAC libraries. For example, in various YAC libraries, a fraction of
the clones result from co-cloning events, i.e., they include in a
single clone noncontiguous DNA fragments. A high percentage of YAC
clones, particularly clones having high molecular weight inserts,
are chimeric. Chimeric clones map to multiple sites on the
chromosome and, thus, hammer the progress of mapping and analysis.
Another problem endemic to YAC cloning is caused by DNA segments
that are unclonable or unstable and tend to rearrange and
delete.
[0007] Bacteria Artificial Chromosomes (BACs), provide an
alternative to the YAC system. BACs mitigate the most problematic
aspects of YACs such as, for example the high rate of chimerism and
clonal instability. BACs are based on the E. coli single-copy
plasmid F factor and are capable of faithful propagation of DNA
fragments greater than about 300 Kb in size. BACs have a number of
physical properties that make them amenable to physical mapping,
including easy manipulation and an absence of chimerism. The lack
of chimerism and the capacity to propagate large exogenous insert
DNAs make the BACs excellent candidates for chromosome walking and
the generation of contiguous physical maps.
[0008] The need for molecular description of chromosome 21 derives
directly from the association with several human genetic diseases.
A map of contiguous units (contigs) covering this chromosome will
speed the identification of the cause of these diseases. Indeed, a
detailed map would provide immediate access to the genomic segmen,
including any pathological locus, as soon as it has been localized
by genetic linkage or cytogenetic analysis.
[0009] Thus, a need exists for identifying, characterizing, and
mapping the numerous genes that occupy loci on chromosome 21, which
will expedite the rapid translation of high resolution chromosome
maps into biological, medical and diagnostic applications. The
present invention satisfies this need and provides related
advantages as well.
SUMMARY OF THE INVENTION
[0010] The present invention provides isolated nucleic acids
encoding human EHOC-1 protein and isolated receptor proteins
encoded thereby. Further provided are vectors containing invention
nucleic acids, probes that hybridize thereto, host cells
transformed therewith, antisense oligonucleotides thereto and
compositions containing, antibodies that specifically bind to
invention polypeptides and compositions containing, as well as
transgenic non-human mammals that express the invention
protein.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1 shows a physical map thr the consensus region for
HPE1.
[0012] FIG. 2 shows a physical map for the consensus region for
EPM1 in relation to the consensus region for HPE1. The locations of
YAC clones, BAC clones and EHOC-1 were indicated by thick bars.
[0013] FIG. 3A shows the regions in which EHOC-1 has homologies to
transmembrane proteins. Region 1 represents 29.4% identity in a 34
amino acid overlap with rat sodium channel protein III. Region 2
represents 20.4% identity in a 103 amino acid overlap with
phosphoglycerate transport system regulatory protein of Salmonella
typhimurin. Region 3 represents 29.1; identity in a 55 amino acid
overlap with pyrophosphate-energized vacuolar membrane proton pump
of Arabidopsis thaliana. Region 4 represents 24.0% identity in a 50
amino acid overlap with myosin-like protein of Saccharomyces
cerevisiae. Region 5 represents 17.9% identity in a 39 amino acid
overlap with rabbit cardiac muscle ryanodine receptor. Region 6
represents 21.0% identity in a 62 amino acid overlap with rat
cardiac muscle sodium channel protein alpha subunit. Region 7
represents 40.7% identity in a 27 amino acid overlap with rat
skeletal muscle sodium channel protein alpha subunit. Region 8
represents a 30.3% identity in a 33 amino acid overlap with
dystrophin cysteine-rich domain.
[0014] FIG. 3B shows a comparison of genetic distances to the EPM1
locus in centiMorgans as computed by linkage diseauilibrium
studies. (Lehesjoki et al., Hum. Mol. Genet. 2:1229-1234
(1993)).
DETAILED DESCRIPTION OF THE INVENTION
[0015] Progressive myoclonus epilepsies (PMEs) are a heterogenous
group of diseases which are characterized by myoclonus, epileptic
seizures and progressive neurological deterioration including
ataxia and dementia Berkovic et al., New Engl. J. Med. 315:296-305
(1986). PME of Unverricht-Lundborg type (EPM1) is an autosomal
recessive disorder with frequent consanguinity in Finland and
Mediterranean regions with the incidence of at least 1: 20,000 in
Finland. Genetic linkage analysis revealed that the locus for EPM1
is on chromosome 21q22.3 Malafosse et al., Lancet 339:1080-1081
(1992) and excluded Lafora disease from this region which is also a
member of PME Lehesjoki et al., Neurology 42:1545-1550 (1992).
Linkage disecuilibrium analysis made it possible to narrow down the
candidate region to 300 kb spanning the loci of PFKL, D21S25 and
D21S154 Lehesjoki et al., Hum. Mol. Genet. 2:1229-1234 (1993);
Lehesjoki et al., Human Genetics 93:668-674 (1994).
[0016] Autoimmune polyglandular disease type 1 (APECED) as also
mapped to chromosome 21q22.3 by linkage disequilibrium analysis
Aaltonen, J., et al., Nature Genet. 8:83-87 (1994). APECED is an
autosomal recessive disease resulting in a variable combination of
failure of the parathyroid glands, adrenal cortex, gonads
pancreatic .beta. cells, thyroid gland and gastric parietal cells.
Additional affects of APECED include alopecia, vitiligo, hepatitis,
chronic mucoccutaneous candidiasis, dystrophy of the dental enamel
and nails and keratopathy. APCED usually manifests itself in
childhood, but tissue specific symptoms may appear throughout
adulthood. The APCED locus maps within 500 kb of D21S49 and
D21S171.
[0017] Holoprosencephaly is characterized by impaired cleavage of
the embryonic forebrain and incomplete mid-facial development that
manifest as a wide range of midfacial anomalies including cyclopia,
ethmocephaly cebocephaly, premaxillary agenesis, hypotelorism, and
a single maxillary central incisor. The most commonly associated
chromosomal abnormaliy includes dup(3p), del(7q), deletions of
chromosome 13, trisomy 13, trisomy 18, and triploidy (Munke, AM J
Med Genet 34:237-245 (1989)). The etiology is heterogeneous and may
include aneuploidies for chromosomes 2, 3, 7, 13, 18 and 21. In
order to narrow down the candidate region for HPE1, the deletion of
21(q22.3) was characterized in two HP patients by fluorescence in
situ hybridization and quantitative Southern blot dosage analysis.
For the smaller deletion, the regions for D21S25, D21S154, D21S171
and D21S44 were deleted and for D21S42 and D21S49 were not.
Combining these data with previous reports of deletion of 21q22.3
(D21S112-ter) without the holoprosencephaly phenotype indicate that
the region responsible for holoprosencephaly spans the 1-2Mb region
including PFKL and ITGB2 (CD18). Four cases of holoprosencephaly
with chromosome 21 anomalies have been published. Estabrooks et al.
describe a minute deletion of chromosome 21(q22.3) (Estabrooks et
al., AM J Med Genet, 36:306-309 (1990)) suggesting this region as a
locus for holoprosencephaly (HPE1).
[0018] Described in the instant specification is the construction
of the BAC (Bacterial Artificial Chromosome) Shizuya et al., Proc.
Natl. Acad. Sci. USA 89:8794-8797 (1992) contig of this
EPM1-APECED-HPE1 candidate region and the isolation of a novel gene
from this contiguous map unit using a direct cDNA selection
technique.
[0019] In order to isolate genes responsible for these diseases, a
cDNA library from a 14-week trisomy 21 fetal brain was constructed
using Uni-Zap XR (Stragene, La. Jolla, Calif.). More than 950
clones have inserts ranging from 1-4 kb (avg. 2 kb). in addition, a
direct cDNA selection method was applied so BACs (Bacterial
Artificial Chromosomes) in the 21q22.3 region. Using cDNA
synthesized from trisomy 21 fetal brain, Sau3AI linkers were
attached, the cDNA then was digested with Sau3AI, followed by
attachment of a second pair of linkers and hybridized o
biotinylated BAC DNAs which cover the candidate region. cDNA/BAC
DNA hybrid molecules were captured on streptavidin coated magnetic
beads, non-specific cDNA were washed out, and specifically
hybridized cDNA were eluted and amplified by PCR. Twice selected
PCR products were subcloned and analyzed. Southern blot analysis
revealed that 21 out of 30 (70%) of fragments yielded unique bands
of the original BACs. Using these fragments as probes, cDNAs (3 kb,
4 kb and 5 kb) were isolated from the library. The 5 kb cDNA
subclone (EHOC-1) maps proximal to but neighboring D21S25 and
showed homologies to transmembrane genes. The loci of these genes
all map within the consensus region where holoprosencephaly, EPM1
and APECED are localized.
[0020] DNA sequence analysis of Skb cDNA showed a complete coding
sequence of 3570 bp which revealed to have homologies to
transmembrane proteins including three kinds of sodium channel
proteins on amino acid sequence level. (SEQ ID NOS: 1-2; FIG.
3).
[0021] Five types of BAC clone were isolated from the total human
genomic DNA BAC library Shizuya et al., Proc. Natl. Acad. Sci. USA
89:8794-8797 (1992) by PCR screening method using STSs containing
PFKL, D21S25, D21S154 and CD18. Physical maps of he
HPE1-EPM1-APECED consensus region with these BAC clones and YAC
clones Chumakov et al., Nature 359:380-387 (1992) is described in
FIGS. 1 and 2. BAC-1 (230 kb) and BAC-2 (210 kb) were positive for
D21S25. BAC-3 (170 kb) was positive for D21S25 and PFKL. Agarose
gel electrophoresis of EcoRI-digested BAC DNAs and Southern blot
analysis showed that these 3 BACs overlap each other. BAC-4 was
identical to BAC-3. BAC-S (100 kb) was positive for CD18.
[0022] Direct cDNA selection was performed on 5 BAC DNAs (four of
which were overlapping) which span the consensus region. EcoRI
digestion of subclone DNAs revealed that 10% clones were chimeric.
The average sizes of the inserts of non-chimeric clones were 400
bp. Forty non-chimeric subclones of selected cDNAs were analyzed by
using EcoRI-digested BAC DNA Southern blots. Twenty-eight clones
(70%) showed unique signals on the BAC blots, 6 clones (15%) showed
repetitive, and 6 clones (15%) did not show any signal on these
blots. Using insert DNAs of these subclones as probes, a trisomy 21
fetal brain cDNA library was screened. Three overlapping cDNAs (3
kb, 4 kb and 5 kb) containing poly (A) tail were isolated and
designated EHOC-1.
[0023] The three overlapping EHOC-1 cDNA subclones were used for
Southern blot analysis using EcoRI-digested BAC DNA blots. Only
BAC-1 showed unique multiple band signals indicating that these
cDNAs originated from BAC-1. Identical sizes of the signal bands
indicated that these clones overlap each other. Complete sequence
of the EHOC-1 5 kb cDNA clone and partial sequence analysis of 3 kb
and 4 kb clones showed that entire sequence of the 3 kb clone and
part of the sequence of 4 kb clone are contained in the 5 kb clone,
but the 3' end of the 4 kb clone was different from that of 5 kb
clone indicating the existence of splice variants of EHOC-1 cDNAs.
Northern blot analysis using the insert of 5 kb EHOC-1 cDNA
revealed three transcripts (5.3 kb, 7.5 kb and 8 kb) on multiple
adult tissues (heart, brain, placenta, lung, liver, skeletal
muscle, kidney, pancreas) Fluorescence in-situ hybridization was
also done on lymphocytes of a normal individual using insert of the
EHOC-1 5 kb cDNA subclone. Discrete signals were seen on chromosome
21q22.3 confirming the loci. The complete sequence of 5 kb cDNA
clone revealed an open reading frame of 3570bp (1190 amino acid).
The initiator ATG was located within a good Kozak consensus
sequence Kozak, M., J. Mol. Biol. 196:947-950 (1987); Kozak, M.,
Nuc. Acid Res. 15:8125-8148 (1987). Homology search of an amino
acid sequence of this ORF to genes registered in Genbank/EMBL
showed that this gene product has homologies to multiple
transmembrane proteins including three types of sodium channel
proteins (FIG. 3).
[0024] Some neurologic disorders in humans are known to result from
mutations in sodium channels Ptacek et al., Cell 67:1021-1027
(1991); Rojas et al., Nature 354:387-389 (1991); McClatchey et al.,
Cell 68:769-774 (1992); Ptacek et al., Neuron 8:891-897 (1992),
calcium channels Ptacek et al., Cell 77: 863-868 (1994);
Jurkut-Rott et al., Hum. Mol. Genet. 3:1415-1419 (1994), and a
potassium channel Browne et al., Nature Genet. 3:136-140 (1994). By
using BLAST computer program Altschul, S.J., et al., J. Mol. Biol.
215:403-410 (1994), one fibronectin domain (CxV . . . YxC) was
found at 356401 a.a. The analysis also showed that the motif
(Sxxx(I,L)E) occurs at 462, 670, 708, 716, 730, and 1078. This
motif was searched for various protein databases and there were
very few where it was present three or more times. These include;
rat cartilage specific proteoglycan core protein, myosin,
drosodhila sevenless (4 copies), drosophila prospero (4 copies),
and drosophila serendipity (3 copies). The latter three are mutants
in development. Sevenless causes an eye defect, prospero defects in
axon pathfinder, and serendipity defects in cellularization. It is
reasonable that a defect in axonal routing may correlate with the
phenotype of EHOC-1. The region beginning at 777 also has some
homologies to multiple drug resistance genes and to the drosophila
rutabaga gene. Rutabaaa is involved in learning in drosophila.
[0025] Accordingly, the present invention provides isolated nucleic
acids encoding a novel gene, EHOC-1, which exists in human
chromosome 21, specifically at the q23.2 locus, which is the site
of mutation(s) that cause PME, HPE1, and APECED. The term "Nucleic
acids" (also referred to as polynucleotides) encompasses RNA as
well as single and double-stranded DNA and cDNA. As used herein,
the phrase "isolated" means a nucleic acid that is in a form that
does not occur in nature. One means of isolating a nucleic acid
encoding an EHOC-1 polypeptide is to probe a mammalian genomic
library with a natural or artificially designed DNA probe using
methods well known in the art. DNA probes derived from the EHOC-1
gene are particularly useful for this purpose. DNA and cDNA
molecules that encode EHOC-1 polypeptides can be used to obtain
complementary genomic DNA, cDNA or RNA from human, mammalian, or
other animal sources, or to isolate related cDNA or genomic clones
by the screening of cDNA or genomic libraries, by methods described
in more detail below. Examples of nucleic acids are RNA, cDNA, or
isolated genomic DNA encoding an EHOC-1 polypeptide. Such nucleic
acids may have coding sequences substantially the same as the
coding sequence shown in SEQ ID NO: 2. This invention also
encompasses nucleic acids which differ from the nucleic acids shown
in SEQ ID NO: 1, but which have the same phenotype, i.e., encode
substantially the same amino acid sequence set forth in SEQ ID NO:
2.
[0026] Phenotypically similar nucleic acids are also referred to as
"functionally equivalent nucleic acids". As used herein, the phrase
"functionally equivalent nucleic acids" encompasses nucleic acids
characterized by slight and non-consequential sequence variations
that will function in substantially the same manner to produce the
same protein product(s) as the nucleic acids disclosed herein. In
particular, functionally equivalent nucleic acids encode
polypeptides that are the same as those disclosed herein or that
have conservative amino acid variations. For example, conservative
variations include substitution of a non-polar residue with another
non-polar residue, or substitution of a charged residue with a
similarly charged residue. These variations include those
recognized by skilled artisans as those that do not substantially
alter the tertiary structure of the protein.
[0027] Further provided are nucleic acids encoding EHOC-1
polypeptides that, by virtue of the degeneracy of the genetic code,
do not necessarily hybridize to the invention nucleic acids under
specified hybridization conditions. Preferred nucleic acids
encoding the invention polypeptide are comprised of nucleotides
that encode substantially the same amino acid sequence set forth in
SEQ ID NO: 2. Alternatively, preferred nucleic acids encoding the
invention polypept4de(s) hybridize under high stringency conditions
to substantially the entire sequence, or substantial portions
(i.e., typically at least 15-30 nucleotides) of the nucleic acid
sequence set forth in SEQ ID NO: 1.
[0028] Stringency of hybridization, as used herein, refers to
conditions under which polynucleotide hybrids are stable. As known
to those of skill in the art, the stability of hybrids is a
function of sodium ion concentration and temperature. (See, for
example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2d
Ed. (Cold Spring Harbor Laboratory, 1989; incorporated herein by
reference)
[0029] Also provided are isolated peptides, polypeptides (s) and/or
protein(s) encoded by the invention nucleic acids which are EHOC-1
polypeptides. The EHOC-1 polypeptide comprises a protein of
approximately 1190 amino acids in length. The predicted amino acid
sequence encoding the EHOC-1 polypeptide is set forth in SEQ ID NO:
2.
[0030] As used herein, the term "isolated" means a protein molecule
free of cellular components and/or contaminants normally associated
with a native in vivo environment. Invention polypeptides and/or
proteins include any natural Occurring allelic variant, as well as
recombinant forms thereof. The EHOC-1 polypeptides can be isolated
using various methods well known to a person of skill in the art.
The methods available for the isolation and purification of
invention proteins include, precipitation, gel filtration,
ion-exchange, reverse-phase and affinity chromatography. Other
well-known methods are described in Deucscher et al., Guide to
Protein Purification: Methods in Enzymology Vol. 182, (Academic
Press, 1990), which is incorporated herein by reference.
Alternatively, the isolated polypeptides of the present invention
can be obtained using well-known recombinant methods as described,
for example, in Sambrook et al., supra., 1989).
[0031] An example of the means for preparing the invention
polypeptides) is to express nucleic acids encoding the EHOC-1 in a
suitable host cell, such as a bacterial cell, a yeast cell, an
amphibian cell (i.e., oocyte), or a mammalian cell, using methods
well known in the art, and recovering the expressed polypeptide,
again using well-known methods. Invention polypeptides can be
isolated directly from cells that have been transformed with
expression vectors, described below in more detail. The invention
polypeptide, biologically active fragments, and functional
equivalents thereof can also be produced by chemical synthesis. As
used herein, "biologically active fragment" refers to any portion
of the polypeptide *represented by the amino acid sequence in SEQ
ID NO: 2 that can assemble into a cationic channel permeable to
Ca.sup.2+ which is activated by acetylcholine. Synthetic
polypeptides can be produced using Applied Biosystems, Inc. Model
430A or 431A automatic peptide synthesizer (Foster City, Calif.)
employing the chemistry provided by the manufacturer.
[0032] As used herein, the phrase "EHOC-1" refers to recombinantly
expressed/produced (i.e., isolated or substantially pure) proteins
that contain highly hydrophobic regions which predict potential
membrane spanning regions, having homologies to multiple
transmembrane proteins, including sodium channel, calcium channel
and potassium channel proteins including variants thereof encoded
by mRNA generated by alternative splicing of a primary transcript,
and further including fragments thereof which retain one or more of
the aforementioned properties. As used herein, the phrase
"functional polypeptide" means that binding of ligands, for
example, cause transcriptional activation of EHOC-1 proteins. More
specifically, agonist activation of a "functional invention
polypeptide" induces the protein to generate a signal.
[0033] Modification of the invention nucleic acids, polypeptides or
proteins with the following phrases: "recombinantly
expressed/produced", "isolated", or "substantially pure",
encompasses nucleic acids, peptides, polypeptides or proteins that
have been produced in such form by the hand of man, and are thus
separated from their native in vivo cellular environment. As a
result of this human intervention, the recombinant nucleic acids,
polypeptides and proteins of the invention are useful in ways that
the corresponding naturally occurring molecules are not, such as
identification of selective drugs or compounds.
[0034] Sequences having "substantial sequence homology" are
intended to refer to nucleotide sequences that share at least about
90% identity with invention nucleic-acids; and amino acid sequences
that typically share at least about 95% amino acid identity with
invention polypeptides. It is recognized, however, that
polypeptides or nucleic acids containing less than the
above-described levels of homology arising as splice variants or
that are modified by conservative amino acid substitutions, or by
substitution of degenerate codons are also encompassed within the
scope of the present invention.
[0035] The present invention provides the isolated polynucleotide
operatively linked to a promoter of RNA transcription, as well as
other regulatory sequences. As used herein, the phrase "operatively
linked" refers to the functional relationship of the polynucleotide
with regulatory and effector sequences of nucleotides, such as
promoters, enhancers, transcriptional and translational stop sites,
and other signal sequences. For example, operative linkage of a
polynucleotide to a promoter refers to the physical and functional
relationship between the polynucleotide and the promoter such that
Transcription of DNA is initiated from the promoter by an RNA
polymerase that specifically recognizes and binds to the promoter,
and wherein the promoter directs the transcription of RNA from the
polynucleotide.
[0036] Promoter regions include specific sequences that are
sufficient for RNA polymerase recognition, binding and
transcription initiation. Additionally, promoter regions include
sequences that modulate the recognition, binding and transcription
initiation activity of RNA polymerase. Such sequences may be cis
acting or may be responsive to trans acting factors. Depending upon
the nature of the regulation, promoters may be constitutive or
regulated. Examples of promoters are SP6, T4, T7, SV40 early
promoter, cytomegalovirus (CMV) promoter, mouse mammary tumor virus
(MMTV) steroid-inducible promoter, Moloney murine leukemia virus
(MMLV) promoter, and the like.
[0037] Vectors that contain both a promoter and a cloning site into
which a polynucleotide can be operatively linked are well known in
the art. Such vectors are capable of transcribing RNA in vitro or
in vivo, and are commercially available from sources such as
Stratagene (La. Jolla, Calif.) and Promega Biotech (Madison, Wis.).
In order to optimize expression and/or in vitro transcription, it
may be necessary to remove, add or alter 5' and/or 3' untranslated
portions of the clones to eliminate extra, potential inappropriate
alternative translation initiation codons or other sequences that
may interfere with or reduce expression, either at the level of
transcription or translation. Alternatively, consensus ribosome
binding sites can be inserted immediately 5' of the start codon to
enhance expression. (See, for example, Kozak, J. Biol. Chem.
266:-9867 (1991)). Similarly, alternative codons, encoding the same
amino acid, can be substituted for coding sequences of the EHOC-1
polypeptide in order to enhance transcription (e.g., the codon
preference of the host cell can be adopted, the presence of G-C
rich domains can be reduced, and the like).
[0038] Also provided are vectors comprising the invention nucleic
acids. Examples of vectors are viruses, such as baculoviruses and
retroviruses, bacteriophages, cosmids, plasmids and other
recombination vehicles typically used in the art. Polynucleotides
are inserted into vector genomes using methods well known in the
art. For example, insert and vector DNA can be contacted, under
suitable conditions, with a restriction enzyme to create
complementary ends on each molecule that can pair with each other
and be joined together with a ligase. Alternatively, synthetic
nucleic acid linkers can be ligated to the termini of restricted
polynucleotide. These synthetic linkers contain nucleic acid
sequences that correspond to a particular restriction site in the
vector DNA. Additionally, an oligonucleotide containing a
termination codon and an appropriate restriction site can be
ligated for insertion into a vector containing, for example, some
or all of the following: a selectable marker gene, such as the
neomycin gene for selection of stable or transient transfectants in
mammalian cells; enhancer/promoter sequences from the immediate
early gene of human CMV for high levels of transcription;
transcription termination and RNA processing signals from SV40 for
mRNA stability; SV40 polyoma origins of replication and ColEl for
proper episomal replication; versatile multiple cloning sites; and
T7 and SP6 RNA promoters for in vitro transcription of sense and
antisense RNA. Other means are well known and available in the
art.
[0039] Also provided are vectors comprising a nucleic acids
encoding an EHOC-1 polypeptide, adapted fo: expression in a
bacterial cell, a yeast cell, an amphibian cell (i.e., oocyte), a
mammalian cell and other animal cells. The vectors additionally
comprise the regulatory elements necessary for expression of the
nucleic acid in the bacterial, yeast, amphibian, mammalian or
animal cells so located relative to the nucleic acid encoding
EHOC-1 polypeptide as to permit expression thereof. As used herein,
"expression" refers to the process by which nucleic acids are
transcribed into mRNA and translated into peptides, polypeptides,
or proteins. If the nucleic acid is derived from genomic DNA,
expression may include splicing of the mRNA, if an appropriate
eucaryotic host is selected. Regulatory elements required for
expression include promoter sequences to bind RNA polymerase and
transcription initiation sequences for ribosome binding. For
example, a bacterial expression vector includes a promoter such as
the lac promoter and for transcription initiation the
Shine-Dalgarno sequence and the start codon AUG (Sambrook et al.
supra). Similarly, a eucaryotic expression vector includes a
heterologous or homologous promoter for RNA polymerase II, a
downstream polyadenylationn signal, the start codon AUG, and a
termination codon for detachment of the ribosome. Such vectors can
be obtained commercially or assembled by the sequences described in
methods well known in the art, for example, the methods described
above for constructing vectors in general. Expression vectors are
useful to produce cells that express the invention polypeptide.
[0040] This invention provides a transformed host cell that
recombinantly expresses the EHOC-1 polypeptide. The host cell has
been transformed with a nucleic acid encoding a EIOC-1 polypeptide.
An example is a mammalian cell comprising a plasmid adapted for
expression in a mammalian cell. The plasmid contains a nucleic acid
encoding an EHOC-1 polypeptide and the regulatory elements
necessary for expression of the invention protein. Various
mammalian cells may be utilized as hosts, including, for example,
mouse fibroblast cell NIH3T3, CHO cells, HeLa cells, Ltk-cells,
etc. Expression plasmids such as those described supra can be used
to transfect mammalian cells by methods well known in the art such
as calcium phosphate precipitation, DEAE-dextran, electroporation,
microinjection or lipofection.
[0041] EHOC-1 polypeptides expressed recombinantly on eucaryotic
cell surfaces may contain at least one EHOC-1 polypeptide, or may
contain a mixture of peptides encoded by the host cell and/or
subunits encoded by heterologous nucleic acids.
[0042] The present invention provides nucleic acid probes
comprising nucleotide sequences capable of specifically hybridizing
with sequences included within the nucleic acid sequence encoding
an EHOC-1 polypeptide, for example, a coding sequence included
within the nucleotide sequence shown in SEQ ID NO: 1. As used
herein, a "probe" is a single-stranded DNA or RNA that has a
sequence of nucleotides that includes at least 15 contiguous bases
set forth in SEQ ID NO: 1. Preferred regions from which to
construct probes include c, and/or 3' coding sequences, sequences
within the ORF, sequences predicted to encode transmembrane
domains, sequences predicted to encode cytoplasmic loops, signal
sequences, ligand binding sites, and the like. Full-length or
fragments of cDNA clones can also be used as probes for the
detection and isolation of related genes. When fragments are used
as probes, preferably the cDNA sequences will be from the carboxyl
end-encoding portion of the cDNA, and most preferably will include
predicted transmembrane domain-encoding portions of the cDNA
sequence. Transmembrane domain regions can be predicted based on
hydropathy analysis of the deduced amino acid sequence using, for
example, the method of Kyte and Doolittle, J. Mol. Biol. 157:105
(1982).
[0043] As used herein, the phrase "specifically hybridizing"
encompasses the ability of a polynucleotide to recognize a sequence
of nucleic acids that are complementary thereto and to form
double-helical segments via hydrogen bonding between complementary
base pairs. Nucleic acid probe technology is well known to those
skilled in the art who will readily appreciate that such probes may
vary greatly in length and may be labeled with a detectable agent,
such as a radioisotope, a fluorescent dye, and the like, to
facilitate detection of the probe. Invention probes are useful to
detect the presence of nucleic acids encoding the EROC-1
polypeptide. For example, the probes can be used for in situ
hybridizations in order to locate biological tissues in which the
invention gene is expressed. Additionally, synthesized
oligonucleotides complementary to the nucleic acids of a nucleotide
sequence encoding EHOC-1 polypeptide are useful as probes for
detecting the invention genes, their associated mRNA, or for the
isolation of related genes using homology screening of genomic or
cDNA libraries, or by using amplification techniques well known to
one of skill in the art.
[0044] Also provided are antisense oligonucleotides having a
sequence capable of binding specifically with any portion of an
mRNA that encodes the EHOC-1 polypeptide so as to prevent
translation of the mRNA. The antisense oligonucleotide may have a
sequence capable of binding specifically with any portion of the
sequence of the cDNA encoding the EHOC-1 polypeptide. As used
herein, the phrase "binding specifically" encompasses the ability
of a nucleic acid sequence to recognize a complementary nucleic
acid sequence and to form double-helical segments therewith via the
formation of hydrogen bonds between the complementary base pairs.
An example of an antisense oligonucleotide is an antisense
oligonucleotide comprising chemical analogs of nucleotides.
[0045] Compositions comprising an amount of the antisense
oligonucleotide, described above, effective to reduce expression of
the EHOC-1 polypeptide by passing through a cell membrane and
binding specifically with mRNA encoding a EHOC-1 polypeptide so as
to prevent its translation and an acceptable hydrophobic carrier
capable of passing through a cell membrane are also provided
herein. The acceptable hydrophobic carrier capable of passing
through cell membranes may also comprise a structure which binds to
a receptor specific for a selected cell type and is thereby taken
up by cells of the selected cell type. The structure may be part of
a protein known to bind to a cell-type specific receptor.
[0046] Antisense oligonucleotide compositions Inhibit translation
of mRNA encoding the invention polypeptides. Synthetic
oligonucleotides, or other antisense chemical structures are
designed to bind to mRNA encoding the EHOC-1 polypeptides and
inhibit translation of mRNA and are useful as compositions to
inhibit expression of EHOC-1 associated genes in a tissue sample or
in a subject.
[0047] This invention provides a means to modulate levels of
expression of EHOC-1 polypeptides by the use of a synthetic
antisense oligonucleotide composition (hereinafter SAOC) which
inhibits translation of mRNA encoding these polypeptides. Synthetic
oligonucleotides, or other antisense chemical structures designed
to recognize and selectively bind to mRNA, are constructed to be
complementary to portions of the nucleotide sequences shown in SEQ
ID NO: 1 of DNA, RNA or chemically modified, artificial nucleic
acids. The SAOC is designed to be stable in the blood stream for
administration to a subject by injection, or in laboratory cell
culture conditions. The SAOC is designed to be capable of passing
through the cell membrane in order to enter the cytoplasm of the
cell by virtue of physical and chemical properties of the SAOC
which render it capable of passing through cell membranes, for
example, by designing small, hydrophobic SAOC chemical structures,
or by virtue of specific transport systems in the cell which
recognize and transport the SAOC into the cell. In addition, the
SAOC can be designed for administration only to certain selected
cell populations by targeting the SAOC to be recognized by specific
cellular uptake mechanisms which bind and take up the SAOC only
within select cell populations. For example, the SAOC may be
designed to bind to a receptor found only in a certain cell type,
as discussed supra. The SAOC is also designed to recognize and
selectively bind to the target mRNA sequence, which may correspond
to a sequence contained within the sequence shown in SEQ ID NO: 1.
The SAOC is designed to inactivate the target mRNA sequence by
either binding to the target mRNA and inducing degradation of the
mRNA by, for example, RNase I digestion, or inhibiting translation
of the mRNA target by interfering with the binding of
transiation-regulating factors or ribosomes, or inclusion of other
chemical structures, such as ribozyme sequences or reactive
chemical groups which either degrade or chemically modify the
target mRNA. SAOCs have been shown to be capable of such properties
when directed against mRNA targets (see Cohen et al., TIPS, 10:435
(1989) and Weintraub, Sci. American, January (1990), pp.40; both
incorporated herein by reference)
[0048] This invention provides a composition containing an
acceptable carrier and any of an isolated, purified EHOC-1
polypeptide, an active fragment thereof, or a purified, mature
protein and active fragments thereof, alone or in combination with
each other. These polypeptides or proteins can be recombinantly
derived, chemically synthesized or purified from native sources. As
used herein, the term "acceptable carrier" encompasses any of the
standard pharmaceutical carries-ems, such as phosphate buffered
saline solution, water and emulsions such as an oil/water or
water/oil emulsion, and various types of wetting agents.
[0049] Also provided are antibodies having specific reactivity with
the EHOC- polypeptides of the subject invention. Active fragments
of antibodies are encompassed within the definition of
"antibody".
[0050] The antibodies of the invention can be produced by methods
known in the art using the invention polypeptides, proteins or
portions thereof as antigens. For example, polyclonal and
monoclonal antibodies can be produced by methods well known in the
art, as described, for example, in Harlow and Lane, Antibodies: A
Laboratory Manual (Cold Spring Harbor Laboratory 1988), which is
incorporated herein by reference. The polypeptide of the present
invention can be used as the immunogen in generating such
antibodies. Alternatively, synthetic peptides can be prepared
(using commercially available synthesizers) and used as nimunogens.
Amino acid sequences can be analyzed by methods well known in the
art to determine whether they encode hydrophobic or hydrophilic
domains of the corresponding polypeptide. Altered antibodies such
as chimer c, humanized, CDR-grafted or bifunctional antibodies an
also be produced by methods well known in the art. Such antibodies
can also be produced by hybridoma, chemical synthesis or
recombinant methods described, for example, in Sambrook et al.,
supra., and Harlow and Lane, supra. Both anti-peptide and
anti-fusion protein antibodies can be used. (see, for example,
Bahouth et al., Trends Pharmacol. Sci. 12:338 (1991); Ausubel et
al., Current Protocols in Molecular Biology (John Wiley and Sons,
N.Y. 1989) which are incorporated herein by reference).
[0051] The invention antibodies can be used to isolate the
invention polypeptides. Additionally the antibodies are useful for
detecting the presence of the invention polypeptides, as well as
analysis of chromosome localization, and structure of functional
domains. Methods for detecting the presence of an EHOC-1
polypeptide on the surface of a cell comprise contacting the cell
with an antibody that specifically binds to the EHOC-1 polypeptide,
under conditions permitting binding of the antibody to the
polypeptides, detecting the presence of the antibody bound to the
cell, and thereby detecting the presence of the invention
polypeptide on the surface of the cell. With respect to the
detection of such polypeptides, the antibodies can be used for in
vitro diagnostic or in vivo imaging methods.
[0052] Immunological procedures useful for in vitro detection of
the target EHOC-1 polypeptide in a sample include immunoassays that
employ a detectable antibody. Such immunoassays include, for
example, ELISA, Pandex microfluorimetric assay, agglutination
assays, flow cytometry, serum diagnostic assays and
immunohistochemical staining procedures which are well known in the
art. An antibody can be made detectable by various means well known
in the art. For example, a detectable marker can be directly or
indirectly attached to the antibody. Useful markers include, for
example, radionucleotides, enzymes, fluorogens, chromogens and
chemiluminescent labels.
[0053] Further, invention antibodies can be used to modulate the
activity of the EHOC-1 polypeptide in living animals, in humans, or
in biological tissues or fluids isolated therefrom. Accordingly,
compositions comprising a carrier and an amount of an antibody
having specificity for the EHOC-1 polypeptide effective to block
binding of naturally occurring ligands to the EHOC-1 polypeptides.
A monoclonal antibody directed To an epitope of an EHOC-1
polypeptide molecule present on the surface of a cell and having an
amino acid sequence substantially the same as an amino acid
sequence for a cell surface epitope of an EHOC-1 polypeptide shown
in SEQ ID NO: 2, can be useful for this purpose.
[0054] The invention provides a transgenic non-human mammal that is
capable of expressing nucleic acids encoding an EHOC-1 polypeptide.
Also provided is a transgenic non-human mammal capable of
expressing nucleic acids encoding an EHOC-1 polypeptide so mutated
as to be incapable of normal activity, i.e., does not express
native EHOC-1. The present invention also provides a transgenic
non-human mammal having a genome comprising antisense nucleic acids
complementary to nucleic acids encoding an EHOC-1 polypeptide so
placed as to be transcribed into antisense mRNA complementary to
mRNA encoding an EHOC-1 polypeptide, which hybridizes thereto and,
thereby, reduces the translation thereof. The nucleic acid may
additionally comprise an inducible promoter and/or tissue specific
regulatory elements, so that expression can be induced, or
restricted to specific cell types. Examples of nucleic acids are
DNA or cDNA having a coding sequence substantially the same as the
coding sequence shown in SEQ ID NO: 1. An example of a non-human
transgenic mammal is a transgenic mouse. Examples of tissue
specificity-determining elements are the metallothionein promoter
and the L7 promoter.
[0055] Animal model systems which elucidate the physiological and
behavioral roles of EHOC-1 polypeptides are produced by creating
transgenic animals in which the expression of the EHOC-1
polypeptide is altered using a variety of techniques. Examples of
such techniques include the insertion of normal or mutant versions
of nucleic acids encoding an EHOC-1 polypeptide by microinjection,
retroviral infection or other means well known to those skilled in
the art, into appropriate fertilized embryos to produce a
transgenic animal. (See, for example, Hogan et al., Manipulating
the Mouse Embryo: A Laboratory Manual (Cold Spring Harbor
Laboratory, 1986). Another technique, homologous recombination of
mutant or normal versions of these genes with the native gene locus
in transgenic animals, may be used to alter the regulation of
expression or the structure of the EHOC-1 polypeptide (see,
Capecchi et al., Science 244:1288 (1989); Zimmer et al., Nature
338:150 (1989); which are incorporated herein by reference).
Homologous recombination techniques are well known in the art.
Homologous recombination replaces the native (endogenous) gene with
a recombinant or mutated gene to produce an animal that cannot
express native (endogenous) protein but can express, for example, a
mutated protein which results in altered expression of the EHOC-1
polypeptide. In contrast to homologous recombination,
microinjection adds genes to the host genome, without removing host
genes. Microinjection can produce a transgenic animal that is
capable of expressing both endogenous and exogenous EHOC-1 protein.
Inducible promoters can be linked to the coding region of the
nucleic acids to provide a means to regulate expression of the
transgene. Tissue specific regulatory elements can be linked to the
coding region to permit tissue-specific expression of the
transaene. Transgenic animal model systems are useful for in vivo
screening of compounds for identification of specific ligands,
i.e., agonists and antagonists, which activate or inhibit protein
responses.
[0056] The nucleic acids, oligonucleotides (including antisense),
vectors containing same, transformed host cells, polypeptides and
combinations thereof, as well as antibodies of the present
invention, can be used to screen compounds in vitro to determine
whether a compound functions as a potential agonist or antagonist,
to the invention polypepo:de. These in vitro screening assays
provide information regarding the function and activity of the
invention polypeptide, which can lead to the identification and
design of compounds that are capable of specific interaction with
one or more types of polypeptides, peptides or proteins.
[0057] In accordance with still another embodiment of the present
invention, there is provided a method for identifying compounds
which bind to EHOC-1 polypeptides. The invention proteins may be
employed in a competitive binding assay. Such an assay can
accommodate the rapid screening of a large number of compounds to
determine which compounds, if any, are capable of binding to EHOC-1
proteins. Subsequently, more detailed assays can be carried out
with those compounds found to bind, to further determine whether
such compounds act as modulators, agonists or antagonists of
invention proteins.
[0058] In another embodiment of the invention, there is provided a
bioassay for identifying compounds which modulate the activity of
invention polypeptides. According to this method, invention
polypeptides are contacted with an "unknown" or test substance (in
the presence of a reporter gene construct when antagonist activity
is tested), the activity of the polypeptide is monitored subsequent
to the contact with the "unknown" or test substance, and those
substances which cause the reporter gene construct to be expressed
are identified as functional ligands for EHOC-1 polypeptides.
[0059] In accordance with another embodiment of the present
invention, transformed host cells that recombinantly express
invention polypeptides can be contacted with a test compound, and
the modulating effect(s) thereof can then be evaluated by comparing
the EHOC-1-mediated response (via reporter gene expression) in the
presence and absence or test compound, or by comparing the response
or test cells or control cells (i.e. , cells that do not express
EHOC-1 polypeptides), to the presence of the compound.
[0060] As used herein, a compound or a signal that "modulates the
activity" of an invention polypeptide refers to a compound or a
signal that alters the activity of EHOC-1 polypeptides so that the
activity of the invention polypeptide is different in the presence
of the compound or signal than in the absence of the compound or
signal. In particular, such compounds or signals include agonists
and antagonists. An agonist encompasses a compound or a signal that
activates EHOC-I protein expression. Alternatively, an antagonist
includes a compound or signal that interferes with EHOC-1 protein
expression. Typically, the effect of an antagonist is observed as a
blocking of agonist-induced protein activation. Antagonists include
competitive and non-competitive antagonists. A competitive
antagonist (or competitive blocker) interacts with or near the site
specific for agonist binding. A non-competitive antagonist or
blocker inactivates the function of the polypeptide by interacting
with a site ocher than the agonist interaction site.
[0061] As understood by those of skill in the art, assay methods
for identifying compounds that modulate EHOC-1 activity generally
require comparison to a control. One type of a "control" is a cell
or culture that is treated substantially the same as the test cell
or test culture exposed to the compound, with the distinction that
the "control" cell or culture is not exposed to the compound. For
example, in methods that use voltage clamp electro physiological
procedures, the same cell can be tested in the presence or absence
of compound, by merely changing the external solution bathing the
cell. Another type of "control" cell or culture may be a cell or
culture that is identical to the transfected cells, with n,e
exception that the "control" cell or culture do not express native
proteins. Accordingly, the response of the transfected cell to
compound is compared to the response (or lack thereof) of the
"control" cell or culture to the same compound under the same
reaction conditions.
[0062] In yet another embodiment of the present invention, the
activation of EHOC-1 polypeptides can be modulated by contacting
the polypeptides with an effective amount of at least one compound
identified by the above-described bioassay.
[0063] The invention will now be described in greater detail with
reference to the following non-limiting examples.
EXAMPLE 1
Construction of BAC Contia
[0064] BAC library construction of total human genomic DNA was
described elsewhere Shizuya et al., Proc. Natl. Acad. Sci. USA
89:8794-8797 (1992). BAC clones were screened by PCR using STSs
(PFKL, D21S25, D21S154, CD18). The loci of these BAC clones were
confirmed bv fluorescence in-situ hybridization. The sizes of
inserts of these clones were measured by running pulsed-field gel
electrophoresis after digesting DNA with NotI.
EXAMPLE 2
Direct cDNA Selection
[0065] Direct selection procedures were similar to those of Morgan
et al. Morgan et al., Nucleic Acid Res. 20:5173-5179 (1992) with
some modifications. Total RNA was isolated from 14 week trisomy 21
fetal brain using TRI regions (Molecular Research Center, Inc.).
Poly (A).sup.+ RNA. was isolated using Poly (A) Quick mRA isolation
kit (STRATAGENE). Double stranded cDNA was synthesized using
uperScript.TM. Choice System (GIBCO BRL) from 5 .mu.g trisomy 21
fetal brain poly (A).sup.+ RNA using 1 .mu.g oligo (dT).sub.15 or
0.1 .mu.g random hexamer. The entire synthesis reaction was
purified by Gene Clean.RTM. II kit (BIO101, Inc.) and was kinased.
Sau3AI linker was attached to cDNA and digested with Sau3AI. The
reaction was purified using Gene Clean. MboI linker I Morgan et
al., Nucleic Acid Res. 20:5173-5179 (1992) was attached to the cDNA
and purified by Gene Clean. Product was amplified by PCR using one
strand of, MboI linker (5' CCTGATGCTCGAG,-AATTC3') as a primer.
Cycling conditions were 40 cycles of 94.degree. C./15 seconds,
60.degree. C./23 seconds, 72.degree. C./2 minutes in a 100 .mu.l of
1.times.PCR buffer (Promega), 3 mM MgCl.sub.2, 5.0 units of Taq
polymerase (Promeaa), 2 .mu.M primer and 0.2 mM dNTPs. Five kinds
of BAC DNA (total 2. .mu.g) was prepared using QIAGEN plasmid kit
and was biotinylated using Nick Translation Kit and biotin-16-dUTP
(Boehringer Manneheim). 3 .mu.g of heat denatured PCR amplified
cDNA was annealed with 3 .mu.g of heat denatured COT1 DNA (BRL) in
100 .mu.l hybridization buffer (750 mM NaCl, 50 mM
NaPO.sub.4(pH7.2), 5 mM EDTA, 5.times.Denhardt's, 0.05% SDS and 50%
formamide) at 42.degree. C. for two hours. After prehybridization,
heat denatured 1.2 .mu.g biotinylated BAC DNA was added and
incubated at 42.degree. C. for 16 hours. cDNA-BAC DNA hybrid was
precipitated with EtOH and dissolved to 60 .mu.l of 10 mM Tris-HCl
(pH 8.0), 1 mM EDTA. After addition of 40 .mu.l 5 M NyaCl, the DNA
was captured on magnetic beads (Dynabeads M-280, Dynal) at
25.degree. C. for 1 hour with gentle rotating. The beads were
washed twice by pipetting in 400 .mu.l of 2.times.SSC, setting in
magnet holder (MPC-E.sub.TM, Dynal) for 30 seconds and removing
supernatant. Four times additional washes were done in
0.2.times.SSC at 68.degree. C. for 10 minutes each with
transferring beads to new tubes at each time. cDNAs were eluted in
100 .mu.l of distilled water for 10 minutes at 80.degree. C. with
occasional mixing. The eluted cDNAs were amplified by PCR as
described above. After repeating selection procedure on magnetic
beads twice, amplified cDNAs were digested with EcoRI and subcloned
into pBluescript II.
EXAMPLE 3
Southern Blot Analysis
[0066] Gel electrophoresis of DNA was carried out on 0.8% agarose
gels in IXTBE. Transfer of nucleic acids to Nybond N+ nylon
membrane (Amersham) was performed by following manufacturer's
instruction. Probes were labelled by RadPrime Labeling System (BRL)
Hybridization was carried out at 42.degree. C. for 16 hours in 50%
formamide, 5.times.SSPE, 5.times. Denhardt's 0.1% SDS, 100 .mu.g/ml
denatured salmon sperm DNA. The filters were washed once in
1.times.SSC, 0.1% SDS at room temperature For 20 minutes, twice in
0.1.times.SSC, 0.1% SDS for 20 minutes at 65.degree. C. Blocs were
exposed to X-ray films (Kodak, X-OMAT-AR).
EXAMPLE 4
cDNA Library Screening
[0067] A trisomy 21 fetal brain cDNA library was constructed using
ZAP-cDNA.RTM. synthesis kit (STRATAGENE) which generates
unidirectional cDNA library. Briefly, double-stranded cDNA was
synthesized from Sg trisomy 21 fetal brain poly(A).sup.+ DINA using
a hybrid oligo(dT)-XhoI linker primer with 5-methyl dCTP, attached
EcoRI linker, digested with EcoRI and XhoI, and cloned into UNI-ZAP
XR vector. The library was packaged using Gigapack.RTM. II Gold
packaging extract. The titer of the original library was
1.1.times.10.sup.6 p.f.u./package. The library was amplified once.
Blue-white color assay indicated that 99% clones have inserts. The
average size of the inserts was 1.9 kb calculated from 14
clones.
[0068] The screening of trisomy 21 fetal brain cDNA library was
performed using selected cDNA fragments. Phages were plated to an
average density of 1.times.10 .sup.5 per 75 cm.sup.2 plate. Plaque
lifts of 20 plates (2.times.10.sup.6 phages) were made using
duplicated nylon membranes (Hybond-N+; Amersham). Hybridized
membranes were washed to final stringency of 0.2.times.SSC,
1.0.times.SDS at 65.degree. C. The filters were exposed to X-ray
film overnight. Phages were subcloned into the plasmid vector
pBluescript II SK(-) by M13-mediated excision for further
analysis.
EXAMPLE 5
Northern Blot Analysis
[0069] cDNA inserts were cut out from the vector by digestion with
XhoI and EcoRI. After labeling using the random priming method, the
fragments were used a probes for Northern hybridization using
Multiple Tissue Northern =lot (Clontech).
EXAMPLE 6
Metaphase Preparation
[0070] Chromosomes were prepared by using a BrdU block, (Zabel et
al. in Proc. Natl. Acad. Sci. USA 80:6932-6936 (1983)) with some
modification. Briefly, human peripheral lymphocytes were grown for
72 hours at 37.degree. C. in RPMI 1640 (GIBCO BRL, Gaithersburg,
Md.) supplemented with -glutamine (2 mM), 15% fetal call serum,
penicillin (100 IU/ml), streptomycin (0.05 mg/ml) and 0.02%
phytohemagglutinin. The cells were blocked n S-phase by adding
5-bromo-deoxyuridine (0.8mg/ml) for 16 hours. They were then washed
once with HBSS (Hanks Balanced Salt Solution) (GIBCO BRL,
Gaithersburg, Md.) to remove the synchronizing agent and were
released by incubating for five to six more hours in medium
supplemented with 2..mu.g/ml of thymidine.
[0071] Cultures were harvested by the addition of 0.1 .mu.g/ml of
colcemid for 10 minutes followed bv 0.075 KCl hypotonic solution
for 15 minutes at 37.degree. C. prior to fixation with a 3:1
mixture of methanol and acetic acid, or 1-5 minutes.
Sequence CWU 0
0
* * * * *