U.S. patent application number 09/859101 was filed with the patent office on 2002-06-06 for novel human dbi/acbp-like protein.
This patent application is currently assigned to Incyte Pharmaceuticals, Inc. Invention is credited to Au-Young, Janice, Goli, Surya K., Hillman, Jennifer L..
Application Number | 20020068825 09/859101 |
Document ID | / |
Family ID | 24814264 |
Filed Date | 2002-06-06 |
United States Patent
Application |
20020068825 |
Kind Code |
A1 |
Au-Young, Janice ; et
al. |
June 6, 2002 |
Novel human DBI/ACBP-like protein
Abstract
The present invention provides polynucleotides which identify
and encode a novel human Diazepam binding inhibitor/acyl-CoA
binding protein (DBI/ACBP)-like protein (DBIH). The invention
provides for genetically engineered expression vectors and host
cells comprising the nucleic acid sequence encoding DBIH. The
invention also provides for the use of substantially purified DBIH
for drug delivery as well as for the production of recombinant
proteins for the treatment of diseases associated with the
expression of DBIH. Additionally, the invention provides for the
use of antisense molecules to DBIH in the treatment of diseases
associated with the expression of DBIH. The invention also
describes diagnostic assays which utilize diagnostic compositions
comprising the polynucleotides which hybridize with naturally
occurring sequences encoding DBIH and antibodies which specifically
bind to the protein.
Inventors: |
Au-Young, Janice; (Berkeley,
CA) ; Hillman, Jennifer L.; (San Jose, CA) ;
Goli, Surya K.; (Sunnyvale, CA) |
Correspondence
Address: |
INCYTE GENOMICS, INC.
PATENT DEPARTMENT
3160 Porter Drive
Palo Alto
CA
94304
US
|
Assignee: |
Incyte Pharmaceuticals, Inc
|
Family ID: |
24814264 |
Appl. No.: |
09/859101 |
Filed: |
May 14, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09859101 |
May 14, 2001 |
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08937823 |
Sep 25, 1997 |
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08937823 |
Sep 25, 1997 |
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08700626 |
Aug 16, 1996 |
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5734038 |
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Current U.S.
Class: |
536/23.7 ;
435/6.14; 435/69.1; 435/7.92; 530/350 |
Current CPC
Class: |
C07K 14/47 20130101;
A61P 25/00 20180101; A61K 38/00 20130101; A61P 43/00 20180101 |
Class at
Publication: |
536/23.7 ;
530/350; 435/69.1; 435/6; 435/7.92 |
International
Class: |
C12Q 001/68; G01N
033/53; G01N 033/537; G01N 033/543; C12P 021/02; C07K 014/705 |
Claims
What is claimed is:
1. An isolated polypeptide comprising an amino acid sequence
selected from the group consisting of: a) a polypeptide comprising
an amino acid sequence of SEQ ID NO: 1, b) a naturally occurring
polypeptide comprising an amino acid sequence at least 90%
identical to an amino acid sequence of SEQ ID NO: 1, c) a
biologically active fragment of a polypeptide having an amino acid
sequence of SEQ ID NO:1, and d) an immunogenic fragment of a
polypeptide having an amino acid sequence of SEQ ID NO:1.
2. An isolated polypeptide of claim 1, having a sequence of SEQ ID
NO:1.
3. An isolated polynucleotide encoding a polypeptide of claim
1.
4. An isolated polynucleotide encoding a polypeptide of claim
2.
5. An isolated polynucleotide of claim 4, having a sequence of SEQ
ID NO:2.
6. A recombinant polynucleotide comprising a promoter sequence
operably linked to a polynucleotide of claim 3.
7. A cell transformed with a recombinant polynucleotide of claim
6.
8. A transgenic organism comprising a recombinant polynucleotide of
claim 6.
9. A method for producing a polypeptide of claim 1, the method
comprising: a) culturing a cell under conditions suitable for
expression of the polypeptide, wherein said cell is transformed
with a recombinant polynucleotide, and said recombinant
polynucleotide comprises a promoter sequence operably linked to a
polynucleotide encoding the polypeptide of claim 1, and b)
recovering the polypeptide so expressed.
10. A method of claim 9, wherein the polypeptide has the sequence
of SEQ ID NO:1.
11. An isolated antibody which specifically binds to a polypeptide
of claim 1.
12. An isolated polynucleotide comprising a sequence selected from
the group consisting of: a) a polynucleotide comprising a
polynucleotide sequence of SEQ ID NO:2, b) a naturally occurring
polynucleotide comprising a polynucleotide sequence at least 90%
identical to a polynucleotide sequence of SEQ ID NO:2, c) a
polynucleotide having a sequence complementary to a polynucleotide
of a), d) a polynucleotide having a sequence complementary to a
polynucleotide of b) and e) an RNA equivalent of a)-d).
13. An isolated polynucleotide comprising at least 60 contiguous
nucleotides of a polynucleotide of claim 12.
14. A method for detecting a target polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide of
claim 12, the method comprising: a) hybridizing the sample with a
probe comprising at least 20 contiguous nucleotides comprising a
sequence complementary to said target polynucleotide in the sample,
and which probe specifically hybridizes to said target
polynucleotide, under conditions whereby a hybridization complex is
formed between said probe and said target polynucleotide or
fragments thereof, and b) detecting the presence or absence of said
hybridization complex, and, optionally, if present, the amount
thereof.
15. A method of claim 14, wherein the probe comprises at least 60
contiguous nucleotides.
16. A method for detecting a target polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide of
claim 12, the method comprising: a) amplifying said target
polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b) detecting the presence or absence of said
amplified target polynucleotide or fragment thereof, and,
optionally, if present, the amount thereof.
17. A composition comprising a polypeptide of claim 1 and a
pharmaceutically acceptable excipient.
18. A composition of claim 17, wherein the polypeptide has an amino
acid sequence of SEQ ID NO:1.
19. A method for treating a disease or condition associated with
decreased expression of functional DBIH, comprising administering
to a patient in need of such treatment the composition of claim
17
20. A method for screening a compound for effectiveness as an
agonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting agonist activity in the sample.
21. A composition comprising an agonist compound identified by a
method of claim 20 and a pharmaceutically acceptable excipient.
22. A method for treating a disease or condition associated with
decreased expression of functional DBIH, comprising administering
to a patient in need of such treatment a composition of claim
21.
23. A method for screening a compound for effectiveness as an
antagonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting antagonist activity in the sample.
24. A composition comprising an antagonist compound identified by a
method of claim 23 and a pharmaceutically acceptable excipient.
25. A method for treating a disease or condition associated with
overexpression of functional DBIH, comprising administering to a
patient in need of such treatment a composition of claim 24.
26. A method of screening for a compound that specifically binds to
the polypeptide of claim 1, said method comprising the steps of: a)
combining the polypeptide of claim 1 with at least one test
compound under suitable conditions, and b) detecting binding of the
polypeptide of claim 1 to the test compound, thereby identifying a
compound that specifically binds to the polypeptide of claim 1.
27. A method of screening for a compound that modulates the
activity of the polypeptide of claim 1, said method comprising: a)
combining the polypeptide of claim 1 with at least one test
compound under conditions permissive for the activity of the
polypeptide of claim 1, b) assessing the activity of the
polypeptide of claim 1 in the presence of the test compound, and c)
comparing the activity of the polypeptide of claim 1 in the
presence of the test compound with the activity of the polypeptide
of claim 1 in the absence of the test compound, wherein a change in
the activity of the polypeptide of claim 1 in the presence of the
test compound is indicative of a compound that modulates the
activity of the polypeptide of claim 1.
28. A method for screening a compound for effectiveness in altering
expression of a target polynucleotide, wherein said target
polynucleotide comprises a polynucleotide sequence of claim 5, the
method comprising: a) exposing a sample comprising the target
polynucleotide to a compound, under conditions suitable for the
expression of the target polynucleotide, b) detecting altered
expression of the target polynucleotide, and c) comparing the
expression of the target polynucleotide in the presence of varying
amounts of the compound and in the absence of the compound.
29. A method for assessing toxicity of a test compound, said method
comprising: a) treating a biological sample containing nucleic
acids with the test compound; b) hybridizing the nucleic acids of
the treated biological sample with a probe comprising at least 20
contiguous nucleotides of a polynucleotide complementary to the
polynucleotide of claim 12, under conditions whereby a specific
hybridization complex is formed between said probe and a target
polynucleotide in the biological sample, said target polynucleotide
comprising a polynucleotide of claim 12; c) quantifying the amount
of hybridization complex; and d) comparing the amount of
hybridization complex in the treated biological sample with the
amount of hybridization complex in an untreated biological sample,
wherein a difference in the amount of hybridization complex in the
treated biological sample is indicative of toxicity of the test
compound.
30. A diagnostic test for a condition or disease associated with
the expression of DBIH in a biological sample comprising the steps
of: a) combining the biological sample with an antibody of claim
11, under conditions suitable for the antibody to bind the
polypeptide and form an antibody:polypeptide complex; and b)
detecting the complex, wherein the presence of the complex
correlates with the presence of the polypeptide in the biological
sample.
31. The antibody of claim 11, wherein the antibody is: a) a
chimeric antibody, b) a single chain antibody, c) a Fab fragment,
d) a F(ab').sub.2 fragment, or e) a humanized antibody.
32. A composition comprising an antibody of claim 11 and an
acceptable excipient.
33. A method of diagnosing a condition or disease associated with
the expression of DBIH in a subject, comprising administering to
said subject an effective amount of the composition of claim
32.
34. A composition of claim 32, wherein the antibody is labeled.
35. A method of diagnosing a condition or disease associated with
the expression of DBIH in a subject, comprising administering to
said subject an effective amount of the composition of claim
34.
36. A method of preparing a polyclonal antibody with the
specificity of the antibody of claim 11 comprising: a) immunizing
an animal with a polypeptide having an amino acid sequence of SEQ
ID NO:1, or an immunogenic fragment thereof, under conditions to
elicit an antibody response; b) isolating antibodies from said
animal; and c) screening the isolated antibodies with the
polypeptide, thereby identifying a polyclonal antibody which binds
specifically to a polypeptide having an amino acid sequence of SEQ
ID NO:1.
37. An antibody produced by a method of claim 36.
38. A composition comprising the antibody of claim 37 and a
suitable carrier.
39. A method of making a monoclonal antibody with the specificity
of the antibody of claim 11 comprising: a) immunizing an animal
with a polypeptide having an amino acid sequence of SEQ ID NO: 1,
or an immunogenic fragment thereof, under conditions to elicit an
antibody response; b) isolating antibody producing cells from the
animal; c) fusing the antibody producing cells with immortalized
cells to form monoclonal antibody-producing hybridomia cells; d)
culturing the hybridoma cells; and e) isolating from the culture
monoclonal antibody which binds specifically to a polypeptide
having an amino acid sequence of SEQ ID NO:1.
40. A monoclonal antibody produced by a method of claim 39.
41. A composition comprising the antibody of claim 40 and a
suitable carrier.
42. The antibody of claim 11, wherein the antibody is produced by
screening a Fab expression library.
43. The antibody of claim 11, wherein the antibody is produced by
screening a recombinant immunoglobulin library.
44. A method for detecting a polypeptide having an amino acid
sequence of SEQ ID NO:1 in a sample, comprising the steps of: a)
incubating the antibody of claim 11 with a sample under conditions
to allow specific binding of the antibody and the polypeptide; and
b) detecting specific binding, wherein specific binding indicates
the presence of a polypeptide having an amino acid sequence of SEQ
ID NO:1 in the sample.
45. A method of purifying a polypeptide having an amino acid
sequence of SEQ ID NO:1 from a sample, the method comprising: a)
incubating the antibody of claim 11 with a sample under conditions
to allow specific binding of the antibody and the polypeptide; and
b) separating the antibody from the sample and obtaining the
purified polypeptide having an amino acid sequence of SEQ ID NO:1.
Description
[0001] This application is a continuation application of U.S.
application Ser. No. 08/937,823 filed Sep. 25, 1997, which is a
divisional of U.S. application Ser. No. 08/700,626, filed Aug. 16,
1996, now U.S. Pat. No. 5,734,038, issued Mar. 31, 1998, entitled
NOVEL HUMAN DBI/ACBP-LIKE PROTEIN, all of which applications and
patents are hereby incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to nucleic acid and amino acid
sequences of a novel human DBI/ACBP-like protein and to the use of
these sequences in the diagnosis, study, prevention and treatment
of disease.
BACKGROUND OF THE INVENTION
[0003] Diazepam binding inhibitor/acyl-CoA binding protein
(DBI/ACBP)-like protein is a 10 kdal protein found in species
ranging from yeast to mammals. It is expressed in a variety of
organs and tissues. Originally, DBI was purified from rat brain
based on its ability to displace diazepam from type A
gamma-aminobutyrate (GAB.sub.A) receptors (Guidotti et al. (1983)
Proc. Natl. Acad. Sci. USA 80:3531-3535). An acyl-Coenzyme A
(acyl-CoA) binding protein (ACBP) subsequently purified from liver
was found to be identical to DBI (Mikkelsen, J. et al. (1987)
Biochem. J. 245:857-861). The protein was known as endozepine, DBI,
or ACBP, but it is now generally referred to as DBI/ACBP. DBI/ACBP,
and polypeptides derived from it, have been implicated in multiple
biological processes, such as 1) GABA.sub.A/benzodiazepam receptor
modulation, 2) acyl-CoA metabolism, 3) steroidogenesis, and 4)
insulin secretion (reviewed in Knudsen J et al (1993) Mol Cell
Biochem 123:129-138).
[0004] The three-dimensional solution structure of bovine DBI/ACBP
with and without bound acyl-CoA ligands has been solved by NMR
(Andersen, K. V. and Poulsen, F. M. (1992) J. Mol. Biol.
226:1131-41; Kragelund et al. (1993) J. Mol. Biol. 230:1260-1277).
DBI/ACBP consists of four alpha helices (A1 through A4) arranged in
a left-handed anti-parallel bundle, with parallel helices A1 and A4
anti-parallel to helices A2 and A3. Helix A2 interacts with each of
the other three helices in a structure reminiscent of a bowl. The
inner surface of the bowl has a patch of non-polar and uncharged
residues at the interface between helices A2 and A3. The rims of
the bowl have mainly polar and charged groups which are contributed
by the hydrophilic residues of the amphipathic helices. The ligand
binding site is located on the inner surface of the bowl, and it
binds the aliphatic acyl chain of the fatty acyl-CoA ligand in a
non-polar arrangement created partly by the protein and partly by
the pantetheine and the adenosine-3'-phosphate of CoA. The
pantetheine and CoA moieties likewise form a highly polar and
charged surface, so that the surface together with the polar and
charged rims of the protein bowl ensure the solubility of the
entire complex (Kragelund et al., supra).
[0005] The binding affinity of bovine DBI/ACBP towards acyl-CoA
esters depends on the length of the acyl chain, where the highest
affinity is for long-chain (C14 to C22) acyl-CoA esters. The
protein is very specific in binding acyl-CoA esters, binding
neither free CoA nor free fatty acids (Rosendal, J. et al. (1993)
Biochem J. 290:321-326). DBI/ACBP sequesters bound long-chain fatty
acyl-CoA, protects acyl-CoAs from hydrolysis, extracts acyl-CoAs
from phosphatidyl choline membranes, and mediates intermembrane
acyl-CoA transport (Rasmussen et al. (1994) Biochem. J.
299:165-170). The overexpression of DBI/ACBP in rapidly growing
brain tumors such as astrocytomas, glioblastomas and
medullablastomas suggests that it may be involved in the regulation
of high-energy acyl-CoA metabolism in rapidly growing neuronal
cells (Alho, H. et al. (1995) Cell. Growth. Differ. 6:309-314).
[0006] DBI/ACBP also inhibits the binding of benzodiazepines to the
GABA.sub.A receptor. The GABA.sub.A receptor is a post-synaptic
Cl.sup.- channel. The Cl.sup.- ion channel opening burst, elicited
by the inhibitory neurotransmitter GABA, is prolonged by
benzodiazepines. Benzodiazepines thereby enhance GABA-mediated
synaptic inhibitory responses and reduce pathological anxiety.
DBI/ACBP or its proteolytic fragments, most notably
octadecaneneuropeptide (ODN, DBI/ACBP amino acids 32-50), suppress
the anxiety-reducing effect of the benzodiazepines. Expression of
DBI/ACBP is increased in brain and cerebrospinal fluid of patients
diagnosed with neurological disorders such as hepatic
encephalopathy, depression and anxiety (Costa, E. and Guidotti, A.
(1991) Life Sciences 49:325-344).
[0007] DBI/ACBP is also involved in the regulation of steroid
biosynthesis in mitochondria (Garnier et al. (1993) Endocrinology
132:444-458). DBI/ACBP stimulates mitochondrial steroidogenesis in
the adrenal gland by facilitating cholesterol delivery to the inner
mitochondrial membrane (Yanagibashi, K. (1988) Endocrinology
123:2075-2082). Knudsen et al. (1993, supra) suggest that DBI/ACBP
may also scavenge fatty acyl-CoA esters produced from fatty acids
released in the conversion of cholesterol to steroids. Antisense
oligonucleotides to DBI/ACBP inhibit hormone-stimulated steroid
production in Leydig cells of rat testis (Boujrad, N. et al. (1993)
Proc. Natl. Acad. Sci. USA 90:5728-5731).
[0008] DBI/ACBP has been found in all tissues tested; the highest
amounts have been found in liver, kidney, brain adrenal gland,
intestine and salivary gland (Knudsen, J. et al., supra).
Immunohistochemical localization indicates DBI/ACBP is selectively
expressed in specialized cells within a given organ. Elevated
levels of DBI/ACBP have been found in cells of the adrenal cortex
and testis which produce steriods, and in liver hepatocytes which
are involved in steriod and fat metabolism. Elevated levels of
DBI/ACBP have also been found in epithelial cells of kidney
tubules, the upper intestinal tracts and large bronchioles, cells
which are specialized for water and electrolyte absorption and
secretion. In brain high DBI/ACBP concentrations are found in
choroid plexus and circumventricular organs, which are specialized
for the control of secretion and osmolality of cerebrospinal fluid
(Bovolin et al. (1990) Reg. Peptides 29:267-281).
[0009] The selective modulation of the expression or activity of a
novel tissue-specific DBI/ACBP-like protein may allow the
successful management of diseases or biochemical abnormalities
relating to the tissues in which it is expressed. In addition, the
binding properties of this small protein may be utilized in drug
delivery applications as a soluble carrier for otherwise insoluble
therapeutic molecules.
SUMMARY OF THE INVENTION
[0010] The present invention discloses a novel tissue-specific
DBI/ACBP-like protein, hereinafter referred to as DBIH, having
chemical and structural homology to isoforms of human DBI/ACBP and
bovine DBI/ACBP. Accordingly, the invention features a
substantially purified DBI/ACBP-like protein, encoded by amino acid
sequence of SEQ ID NO:1, having structural characteristics of the
family of DBI/ACBPs including those from human and cow.
[0011] One aspect of the invention features isolated and
substantially purified polynucleotides which encode DBIH. In a
particular aspect, the polynucleotide is the nucleotide sequence of
SEQ ID NO:2. In addition, the invention features nucleotide
sequences which hybridize under stringent conditions to SEQ ID
NO:2.
[0012] The invention further relates to nucleic acid sequence
encoding DBIH, oligonucleotides, peptide nucleic acids (PNA),
fragments, portions or antisense molecules thereof. The present
invention also relates to an expression vector which includes
polynucleotide encoding DBIH and its use to transform host cells or
organisms. The invention also relates to antibodies which bind
specifically to the DBI of SEQ ID NO:1 and to a pharmaceutical
composition comprising a substantially purified DBI of SEQ ID
NO:1.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIGS. 1A, 1B and 1C show the amino acid sequence (SEQ ID
NO:1) and the nucleic acid sequence (SEQ ID NO:2) of the human
DBI/ACBP-like protein DBIH, produced using MacDNAsis software
(Hitachi Software Engineering Co Ltd, San Bruno Calif.).
[0014] FIGS. 2A and 2B show the amino acid sequence alignments
among DBIH (SEQ ID NO:1), the 104 amino acid human DBI/ACBP isoform
(GI 181478; SEQ ID NO:3), the 86 amino acid human DBI/ACBP isoform
(GI 118276; SEQ ID NO:4), and the 86 amino acid bovine DBI/ACBP (GI
118275, SEQ ID NO:5), produced using the multisequence alignment
program of DNAStar software (DNAStar Inc, Madison Wis.).
[0015] FIG. 3 shows the hydrophobicity plot (generated using
MacDNAsis software) for DBIH, SEQ ID NO:1; the X axis reflects
amino acid position, and the negative Y axis, hydrophobicity.
[0016] FIG. 4 shows the predicted secondary structure (generated
using MacDNAsis software) of DBIH, SEQ ID NO:1.
DETAILED DESCRIPTION OF THE INVENTION
[0017] Definitions
[0018] "Nucleic acid sequence" as used herein refers to an
oligonucleotide, nucleotide or polynucleotide, and fragments or
portions thereof, and to DNA or RNA of genomic or synthetic origin
which may be single- or double-stranded, and represent the sense or
antisense strand. Similarly, amino acid sequence as used herein
refers to peptide or protein sequence.
[0019] "Peptide nucleic acid" as used herein refers to a molecule
which comprises an oligomer to which an amino acid residue, such as
lysine, and an amino group have been added. These small molecules,
also designated anti-gene agents, stop transcript elongation by
binding to their complementary (template) strand of nucleic acid
(Nielsen, P. E. et al. (1993) Anticancer Drug Des. 8:53-63).
[0020] A "variant" of DBIH is defined as an amino acid sequence
that is different by one or more amino acid substitutions. The
variant may have "conservative" changes, wherein a substituted
amino acid has similar structural or chemical properties, eg,
replacement of leucine with isoleucine. More rarely, a variant may
have "nonconservative" changes, eg, replacement of a glycine with a
tryptophan. Similar minor variations may also include amino acid
deletions or insertions, or both. Guidance in determining which and
how many amino acid residues may be substituted, inserted or
deleted without abolishing biological or immunological activity may
be found using computer programs well known in the art, for
example, DNAStar software.
[0021] A "deletion" is defined as a change in either nucleotide or
amino acid sequence in which one or more nucleotides or amino acid
residues, respectively, are absent.
[0022] An "insertion" or "addition" is that change in a nucleotide
or amino acid sequence which has resulted in the addition of one or
more nucleotides or amino acid residues, respectively, as compared
to the naturally occurring DBIH.
[0023] A "substitution" results from the replacement of one or more
nucleotides or amino acids by different nucleotides or amino acids,
respectively.
[0024] The term "biologically active" refers to a DBIH having
structural, regulatory or biochemical functions of the naturally
occurring DBIH. Likewise, "immunologically active" defines the
capability of the natural, recombinant or synthetic DBIH, or any
oligopeptide thereof, to induce a specific immune response in
appropriate animals or cells and to bind with specific
antibodies.
[0025] The term "derivative" as used herein refers to the chemical
modification of a nucleic acid encoding DBIH or the encoded DBIH.
Illustrative of such modifications would be replacement of hydrogen
by an alkyl, acyl, or amino group. A nucleic acid derivative would
encode a polypeptide which retains essential biological
characteristics of natural DBIH.
[0026] As used herein, the term "substantially purified" refers to
molecules, either nucleic or amino acid sequences, that are removed
from their natural environment, isolated or separated, and are at
least 60% free, preferably 75% free, and most preferably 90% free
from other components with which they are naturally associated.
[0027] "Stringency" typically occurs in a range from about
Tm-5.degree. C. (5.degree. C. below the Tm of the probe) to about
20.degree. C. to 25.degree. C. below Tm As will be understood by
those of skill in the art, a stringency hybridization can be used
to identify or detect identical polynucleotide sequences or to
identify or detect similar or related polynucleotide sequences.
[0028] The term "hybridization" as used herein shall include "any
process by which a strand of nucleic acid joins with a
complementary strand through base pairing" (Coombs, J, (1994)
Dictionary of Biotechnology, Stockton Press, New York N.Y.).
Amplification as carried out in the polymerase chain reaction
technologies is described in Dieffenbach, C. W. and G. S. Dveksler
(1995, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press,
Plainview N.Y.).
[0029] Description
[0030] The present invention relates to a novel human DBI/ACBP-like
protein, designated DBIH, initially identified among the partial
cDNAs from a human paraganglia tissue library (PGANNOT01) and to
the use of the nucleic acid and amino acid sequences disclosed
herein in the study, diagnosis, prevention and treatment of
disease. Northern analysis using the LIFESEQ database (Incyte
Pharmaceuticals, Palo Alto Calif.) indicates that DBIH-encoding
mRNA is only present in paraganglia, in contrast to the wide tissue
distribution of the DBI/ACBPs. Paraganglia contain cells that
synthesize, store and secrete catecholamines. Cells of the
paraganglia receive sympathetic preganglionic innervation similar
to that of chromaffin cells of the adrenal medulla. The paraganglia
are well vascularized and the secretory cells are generally located
next to capillaries. With little obstruction to the passage of
hormones, paraganglial cells have both remote and local endocrine
effects.
[0031] The present invention also encompasses DBIH variants. A
preferred DBIH variant is one having at least 80% amino acid
sequence similarity to the DBIH amino acid sequence (SEQ ID NO:1),
a more preferred DBIH variant is one having at least 90% amino acid
sequence similarity to SEQ ID NO: 1 and a most preferred DBIH
variant is one having at least 95% amino acid sequence similarity
to SEQ ID NO: 1.
[0032] The nucleic acid sequence encoding a portion of DBIH was
first identified in the cDNA, Incyte Clone 620984, through a
computer-generated search for amino acid sequence alignments. The
nucleic acid sequence, SEQ ID NO:2, disclosed herein (FIGS. 1A, 1B
and 1C) encodes the amino acid sequence, SEQ ID NO:1, designated
DBIH. The present invention is based in part on the structural
homology shown in FIGS. 2a and 2B, among DBIH and other DBI/ACBPs
including two human isoforms (GI 181478; Gray, P. W. et al. (1986)
Proc. Natl. Acad. Sci. USA 83:7547-7551 and GI 118276; Marquardt,
H. et al. (1986) J. Biol. Chem. 261:9727-9731), and one bovine
isoform (GI 118275, Marquardt, H. et al., supra). GI 181478, GI
118276, and GI 118275 have, respectively, 39%, 42% and 40% sequence
identity with DIBH.
[0033] DBIH consists of 145 amino acids and, based on the
hydropathy plot (FIG. 3) and the secondary structure prediction
(FIG. 4), is a soluble protein consisting of at least four helical
segments. These helices may form an anti-parallel bowl-shaped array
which defines the class of small ligand-binding proteins including
the DBI/ACBPs (Kragelund et al., supra). From its homology to the
central portion of human and bovine DBI/ACBPs, the four
alpha-helices of DBIH are predicted to include residues at or near
positions 45-56, 61-77, 92-103, and 106-121. In comparison with the
86 residue human and bovine DBI/ACBPs (GI 118276 and GI 118275,
respectively), DBIH contains an additional 40 amino acids at the
N-terminus and an additional 14 amino acids near the C-terminus
(FIGS. 2A and 2B). The 104 amino acid human DBI/ACBP isoform (GI
181478) likewise contains an additional 23 amino acids near the
N-terminus compared to the 86 residue forms. The additional
residues in DBIH at the N- and C-termini do not have significant
alpha-helical content (FIG. 4), and contain a large proportion of
hydrophilic charged residues, indicating they are not likely to be
membrane-spanning or signal sequences. However, the additional
residues at the N- or C-termini of DBIH may be proteolytically
processed to yield smaller forms of DBIH in vivo.
[0034] The DBIH Coding Sequences
[0035] The nucleic acid and amino acid sequences of DBIH are shown
in FIGS. 1A, 1B and 1C. In accordance with the invention, any
nucleic acid sequence which encodes the amino acid sequence of DBIH
can be used to generate recombinant molecules which express DBIH.
In a specific embodiment described herein, a partial sequence of
DBIH was first isolated as Incyte Clone 620984 from a human
paraganglia tissue library (PGANNOT01).
[0036] It will be appreciated by those skilled in the art that as a
result of the degeneracy of the genetic code, a multitude of
DBIH-encoding nucleotide sequences, some bearing minimal homology
to the nucleotide sequences of any known and naturally occurring
gene may be produced. The invention contemplates each and every
possible variation of nucleotide sequence that could be made by
selecting combinations based on possible codon choices. These
combinations are made in accordance with the standard triplet
genetic code as applied to the nucleotide sequence of naturally
occurring DBIH, and all such variations are to be considered as
being specifically disclosed.
[0037] Although nucleotide sequences which encode DBIH and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring DBIH under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding DBIH or its derivatives
possessing a substantially different codon usage. Codons may be
selected to increase the rate at which expression of the peptide
occurs in a particular prokaryotic or eukaryotic expression host in
accordance with the frequency with which particular codons are
utilized by the host. Other reasons for substantially altering the
nucleotide sequence encoding DBIH and its derivatives without
altering the encoded amino acid sequences include the production of
RNA transcripts having more desirable properties, such as a greater
half-life, than transcripts produced from the naturally occurring
sequence.
[0038] It is now possible to produce a DNA sequence, or portions
thereof, encoding a DBIH and its derivatives entirely by synthetic
chemistry, after which the synthetic gene may be inserted into any
of the many available DNA vectors and cell systeis using reagents
that are well known in the art at the time of the filing of this
application. Moreover, synthetic chemistry may be used to introduce
mutations into a gene encoding DBIH.
[0039] Also included within the scope of the present invention are
polynucleotide sequences that are capable of hybridizing to the
nucleotide sequence of FIGS. 1A, 1B and 1C under various conditions
of stringency. Hybridization conditions are based on the melting
temperature (Tm) of the nucleic acid binding complex or probe, as
taught in Berger and Kimmel (1987, Guide to Molecular Cloning
Techniques, Methods in Enzvmology, Vol 152, Academic Press, San
Diego Calif.) incorporated herein by reference, and confer may be
used at a defined stringency.
[0040] Altered nucleic acid sequences encoding DBIH which may be
used in accordance with the invention include deletions, insertions
or substitutions of different nucleotides resulting in a
polynucleotide that encodes the same or a functionally equivalent
DBIH. The protein may also show deletions, insertions or
substitutions of amino acid residues which produce a silent change
and result in a functionally equivalent DBIH. Deliberate amino acid
substitutions may be made on the basis of similarity in polarity,
charge, solubility, hydrophobicity, hydrophilicity, and/or the
amphipathic nature of the residues as long as the biological
activity of DBIH is retained. For example, negatively charged amino
acids include aspartic acid and glutamic acid; positively charged
amino acids include lysine and arginine; and amino acids with
uncharged polar head groups having similar hydrophilicity values
include leucine, isoleucine, valine; glycine, alanine; asparagine,
glutamine; serine, threonine phenylalanine, and tyrosine.
[0041] Included within the scope of the present invention are
alleles of DBIH. As used herein, an "allele" or "allelic sequence"
is an alternative form of DBIH. Alleles result from a mutation,
i.e., a change in the nucleic acid sequence, and generally produce
altered mRNAs or polypeptides whose structure or function may or
may not be altered. Any given gene may have none, one or many
allelic forms. Common mutational changes which give rise to alleles
are generally ascribed to natural deletions, additions or
substitutions of amino acids. Each of these types of changes may
occur alone, or in combination with the others, one or more times
in a given sequence.
[0042] Methods for DNA sequencing are well known in the art and
employ such enzymes as the Klenow fragment of DNA polymerase I,
SEQUENASE (US Biochemical Corp, Cleveland Ohio), Taq polymerase
(Perkin Elmer, Norwalk Conn.), thermostable T7 polymerase
(Amersham, Chicago Ill.), or conbinations of recombinant
polymerases and proofreading exonucleases such as the ELONGASE
Amplification System marketed by Gibco BRL (Gaithersburg Md.).
[0043] Preferably, the process is automated with machines such as
the Hamilton Micro Lab 2200 (Hamilton, Reno Nev.), Peltier Thermal
Cycler (PTC200; MJ Research, Watertown Mass.) and the ABI 377 DNA
sequencers (Perkin Elmer).
[0044] Extending the Polynucleotide Sequence
[0045] The polynucleotide sequence encoding DBIH may be extended
utilizing partial nucleotide sequence and various methods known in
the art to detect upstream sequences such as promoters and
regulatory elements. Gobinda et al. (1993; PCR Methods Applic.
2:318-22) disclose "restriction-site" polymerase chain reaction
(PCR) as a direct method which uses universal primers to retrieve
unknown sequence adjacent to a known locus. First, genomic DNA is
amplified in the presence of primer to a linker sequence and a
primer specific to the known region. The amplified sequences are
subjected to a second round of PCR with the same linker primer and
another specific primer internal to the first one. Products of each
round of PCR are transcribed with an appropriate RNA polymerase and
sequenced using reverse transcriptase.
[0046] Inverse PCR can be used to amplify or extend sequences using
divergent primers based on a known region (Triglia, T. et al.
(1988) Nucleic Acids Res. 16:8186). The primers may be designed
using OLIGO 4.06 Primer Analysis Software (1992; National
Biosciences Inc, Plymouth Minn.), or another appropriate program,
to be 22-30 nucleotides in length, to have a GC content of 50% or
more, and to anneal to the target sequence at temperatures about
68.degree.-72.degree. C. The method uses several restriction
enzymes to generate a suitable fragment in the known region of a
gene. The fragment is then circularized by intramolecular ligation
and used as a PCR template.
[0047] Capture PCR (Lagerstroni, M. et al. (1991) PCR Methods
Applic. 1:111-19) is a method for PCR amplification of DNA
fragments adjacent to a known sequence in human and yeast
artificial chromosome DNA. Capture PCR also requires multiple
restriction enzyme digestions and ligations to place an engineered
double-stranded sequence into an unknown portion of the DNA
molecule before PCR.
[0048] Another method which may be used to retrieve unknown
sequences is that of Parker, J. D. et al. (1991; Nucleic Acids Res.
19:3055-60). Additionally, one can use PCR, nested primers and
PromoterFinder libraries to walk in genomic DNA (PROMOTERFINDER,
Clontech (Palo Alto Calif.). This process avoids the need to screen
libraries and is useful in finding intron/exon junctions.
[0049] Preferred libraries for screening for full length cDNAs are
ones that have been size-selected to include larger cDNAs. Also,
random primed libraries are preferred in that they will contain
more sequences which contain the 5' and upstream regions of genes.
A randomly primed library may be particularly useful if an oligo
d(T) library does not yield a full-length cDNA. Genonic libraries
are useful for extension into the 5' nontranslated regulatory
region.
[0050] Capillary electrophoresis may be used to analyze the size or
confirm the nucleotide sequence of sequencing or PCR products.
Systems for rapid sequencing are available from Perkin Elmer,
Beckman Instruments (Fullerton Calif.), and other companies.
Capillary sequencing may employ flowable polymers for
electrophoretic separation, four different fluorescent dyes (one
for each nucleotide) which are laser activated, and detection of
the emitted wavelengths by a charge coupled device camera.
Output/light intensity is converted to electrical signal using
appropriate software (eg. GENOTYPER and SEQUENCE NAVIGATOR from
Perkin Elmer) and the entire process from loading of samples to
computer analysis and electronic data display is computer
controlled. Capillary electrophoresis is particularly suited to the
sequencing of small pieces of DNA which might be present in limited
amounts in a particular sample. The reproducible sequencing of up
to 350 bp of M13 phage DNA in 30 min has been reported
(Ruiz-Martinez, M. C. et al. (1993) Anal. Chem 65:2851-8).
[0051] Expression of the Nucleotide Sequence
[0052] In accordance with the present invention, polynucleotide
sequences which encode DBIH, fragments of the polypeptide, fusion
proteins or functional equivalents thereof may be used in
recombinant DNA molecules that direct the expression of DBIH in
appropriate host cells. Due to the inherent degeneracy of the
genetic code, other DNA sequences which encode substantially the
same or a functionally equivalent amino acid sequence, may be used
to clone and express DBIH. As will be understood by those of skill
in the art, it may be advantageous to produce DBIH-encoding
nucleotide sequences possessing non-naturally occurring codons.
Codons preferred by a particular prokaryotic or eukaryotic host
(Murray, E. et al. (1989) Nuc. Acids Res. 17:477-508) can be
selected, for example, to increase the rate of DBIH expression or
to produce recombinant RNA transcripts having desirable properties,
such as a longer half-life, than transcripts produced from
naturally occurring sequence.
[0053] The nucleotide sequences of the present invention can be
engineered in order to alter a coding sequence of DBIH for a
variety of reasons, including but not limited to, alterations which
modify the cloning, processing and/or expression of the gene
product. For example, mutations may be introduced using techniques
which are well known in the art, eg, site-directed mutagenesis to
insert new restriction sites, to alter glycosylation patterns, to
change codon preference, to produce splice variants, etc.
[0054] In another embodiment of the invention, a natural, modified
or recombinant nucleotide sequence encoding DBIH may be ligated to
a heterologous sequence to encode a fusion protein. For example,
for screening of peptide libraries for inhibitors of DBIH activity,
it may be useful to encode a chimeric DBIH protein that is
recognized by a commercially available antibody. A fusion protein
may also be engineered to contain a cleavage site located between a
DBIH sequence and the heterologous protein sequence, so that the
DBIH may be cleaved and substantially purified away from the
heterologous moiety.
[0055] In an alternate embodiment of the invention, the coding
sequence for DBIH may be synthesized, whole or in part, using
chemical methods well known in the art (see Caruthers, M. H. et al.
(1980) Nuc. Acids Res. Symp. Ser. 215-23, Horn, T, et al, (1980)
Nuc. Acids Res. Symp. Ser. 225-32, etc). Alternatively, the protein
itself could be produced using chemical methods to synthesize a
DBIH amino acid sequence, whole or in part. For example, peptide
synthesis can be performed using various solid-phase techniques
(Roberge, J. Y. et al. (1995) Science 269:202-204) and automated
synthesis may be achieved using the ABI 431A Peptide Synthesizer
(Perkin Elmer) in accordance with the instructions provided by the
manufacturer.
[0056] The newly synthesized peptide can be substantially purified
by preparative high performance liquid chromatography (e.g.,
Creighton (1983) Proteins, Structures and Molecular Principles, WH
Freeman and Co, New York N.Y.). The composition of the synthetic
peptides may be confirmed by amino acid analysis or sequencing
(e.g., the Edman degradation procedure; Creighton, supra).
Additionally the amino acid sequence of DBIH, or any part thereof,
may be altered during direct synthesis and/or combined using
chemical methods with sequences from other proteins, or any part
thereof, to produce a variant polypeptide.
[0057] Expression Systems
[0058] In order to express a biologically active DBIH, the
nucleotide sequence encoding DBIH or its functional equivalent, is
inserted into an appropriate expression vector, ie, a vector which
contains the necessary elements for the transcription and
translation of the inserted coding sequence.
[0059] Methods which are well known to those skilled in the art can
be used to construct expression vectors containing a DBIH coding
sequence and appropriate transcriptional or translational controls.
These methods include in vitro recombinant DNA techniques,
synthetic techniques and in vivo recombination or genetic
recombination. Such techniques are described in Sambrook et al.
(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Press, Plainview N.Y. and Ausubel, F. M. et al. (1989) Current
Protocols in Molecular Biology, John Wiley & Sons, New York
N.Y.
[0060] A variety of expression vector/host systems may be utilized
to contain and express a DBIH coding sequence. These include but
are not limited to microorganisms such as bacteria transformed with
recombinant bacteriophage, plasmid or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with virus expression vectors (eg,
baculovirus); plant cell systems transfected with virus expression
vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus,
TMV) or transformed with bacterial expression vectors (eg, Ti or
pBR322 plasmid); or animal cell systems.
[0061] The "control elements" or "regulatory sequences" of these
systems vary in their strength and specificities and are those
nontranslated regions of the vector, enhancers, promoters, and 3'
untranslated regions, which interact with host cellular proteins to
carry out transcription and translation. Depending on the vector
system and host utilized, any number of suitable transcription and
translation elements, including constitutive and inducible
promoters, may be used. For example, when cloning in bacterial
systems, inducible promoters such as the hybrid lacZ promoter of
the BLUESCRIPT phagemid (Stratagene, LaJolla Calif.) or pSport1
(Gibco BRL) and ptrp4ac hybrids and the like may be used. The
baculovirus polyhedrin promoter may be used in insect cells.
Promoters or enhancers derived from the genomes of plant cells (eg,
heat shock, RUBIS CO; and storage protein genes) or from plant
viruses (eg, viral promoters or leader sequences) may be cloned
into the vector. In mammalian cell systems, promoters from the
mammalian genes or from mammalian viruses are most appropriate. If
it is necessary to generate a cell line that contains multiple
copies of DBIH, vectors based on SV40 or EBV may be used with an
appropriate selectable marker.
[0062] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for DBIH. For example,
when large quantities of DBIH are needed for the induction of
antibodies, vectors which direct high level expression of fusion
proteins that are readily purified may be desirable. Such vectors
include, but are not limited to, the multifunctional E. coli
cloning and expression vectors such as BLUESCRIPT (Stratagene), in
which the DBIH coding sequence may be ligated into the vector in
frame with sequences for the amino-terminal Met and the subsequent
7 residues of .beta.-galactosidase so that a hybrid protein is
produced; pIN vectors (Van Heeke & Schuster (1989) J. Biol.
Chem. 264:5503-5509); and the like. pGEX vectors (Promega, Madison
Wis.) may also be used to express foreign polypeptides as fusion
proteins with glutathione S-transferase (GST). In general, such
fusion proteins are soluble and can easily be purified from lysed
cells by adsorption to glutathione-agarose beads followed by
elution in the presence of free glutathione. Proteins made in such
systems are designed to include heparin, thrombin or factor XA
protease cleavage sites so that the cloned polypeptide of interest
can be released from the GST moiety at will.
[0063] In the yeast, Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters such as alpha
factor, alcohol oxidase and PGH may be used. For reviews, see
Ausubel et al. (supra) and Grant et al. (1987) Methods in
Enzymology 153:516-544.
[0064] In cases where plant expression vectors are used, the
expression of a sequence encoding DBIH may be driven by any of a
number of promoters. For example, viral promoters such as the 35S
and 19S promoters of CaMV (Brisson et al. (1984) Nature
310:511-514) maybe used alone or in combination with the omega
leader sequence from TMV (Takamatsu et al. (1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small
subunit of RUBISCO (Coruzzi et al. (1984) EMBO J. 3:1671-1680;
Broglie et al. (1984) Science 224:838-843); or heat shock promoters
(Winter, J. and Sinibaldi, R. M. (1991) Results Probl. Cell.
Differ. 17:85-105) maybe used. These constructs can be introduced
into plant cells by direct DNA transformation or pathogen mediated
transfection. For reviews of such techniques, see Hobbs, S. or
Murry, L. E. in McGraw Hill Yearbook of Science and Technology
(1992) McGraw Hill New York N.Y., pp 191-196 or Weissbach and
Weissbach (1988) Methods for Plant Molecular Biology, Academic
Press, New York N.Y., pp 421-463.
[0065] An alternative expression system which could be used to
express DBIH is an insect system In one such system Autographa
califonica nuclear polyhedrosis virus (AcNPV) is used as a vector
to express foreign genes in Spodoptera frugiperda cells or in
Trichoplusia larvae. The DBIH coding sequence may be cloned into a
nonessential region of the virus, such as the polyhedrin gene, and
placed under control of the polyhedrin promoter. Successful
insertion of the DBIH coding sequence will render the polyhedrin
gene inactive and produce recombinant virus lacking coat protein
coat. The recombinant viruses are then used to infect S. frugiperda
cells or Trichoplusia larvae in which DBIH is expressed (Smith et
al. (1983) J. Virol. 46:584; Engelhard, E. K. et al. (1994) Proc.
Natl. Acad. Sci. USA 91:3224-7).
[0066] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, a coding sequence for DBIH may be ligated into
an adenovirus transcription/translation complex consisting of the
late promoter and tripartite leader sequence. Insertion in a
nonessential E1 or E3 region of the viral genome will result in a
viable virus capable of expressing DBIH in infected host cells
(Logan and Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-59). In
addition, transcription enhancers, such as the rous sarcoma virus
(RSV) enhancer, may be used to increase expression in mammalian
host cells.
[0067] Specific initiation signals may also be required for
efficient translation of a DBIH sequence. These signals include the
ATG initiation codon and adjacent sequences. In cases where nucleic
acid encoding DBIH, its initiation codon and upstream sequences are
inserted into the appropriate expression vector, no additional
translational control signals may be needed. However, in cases
where only coding sequence, or a portion thereof, is inserted,
exogenous transcriptional control signals including the ATG
initiation codon must be provided. Furthermore, the initiation
codon must be in the correct reading frame to ensure transcription
of the entire insert. Exogenous transcriptional elements and
initiation codons can be of various origins, both natural and
synthetic. The efficiency of expression may be enhanced by the
inclusion of enhancers appropriate to the cell system in use
(Scharf,. D. et al. (1994) Results. Probl. Cell. Differ. 20:125-62;
Bittner et al. (1987) Methods in Enzymol 153:516-544).
[0068] In addition, a host cell strain may be chosen for its
ability to modulate the expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be important for
correct insertion, folding and/or function. Different host cells
such as CHO, HeLa, MDCK, 293, W138, etc have specific cellular
machinery and characteristic mechanisms for such post-translational
activities and may be chosen to ensure the correct modification and
processing of the introduced, foreign protein.
[0069] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express DBIH may be transformed using expression
vectors which contain viral origins of replication or endogenous
expression elements and a selectable marker gene. Following the
introduction of the vector, cells may be allowed to grow for 1-2
days in an enriched media before they are switched to selective
media. The purpose of the selectable marker is to confer resistance
to selection, and its presence allows growth and recovery of cells
which successfully express the introduced sequences. Resistant
clumps of stably transformed cells can be proliferated using tissue
culture techniques appropriate to the cell type.
[0070] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase (Wigler, M. et al. (1977)
Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et
al. (1980) Cell 22:817-23) genes which can be employed in tk- or
aprt-cells, respectively. Also, antimetabolite, antibiotic or
herbicide resistance can be used as the basis for selection; for
example, dhfr which confers resistance to methotrexate (Wigler, M.
et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-70); npt, which
confers resistance to the aminoglycosides neomycin and G-418
(Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14) and als
or pat, which confer resistance to chlorsulfuron and
phosphinotricin acetyltransferase, respectively (Murry, supra).
Additional selectable genes have been described, for example, trpB,
which allows cells to utilize indole in place of tryptophan, or
hisD, which allows cells to utilize histinol in place of histidine
(Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci.
USA 85:8047-51). Recently, the use of visible markers has gained
popularity with such markers as anthocyanins, .beta. glucuronidase
and its substrate, GUS, and luciferase and its substrate,
luciferin, being widely used not only to identify transformants,
but also to quantify the amount of transient or stable protein
expression attributable to a specific vector system (Rhodes, C. A.
et al. (1995) Methods Mol. Biol. 55:121-131).
[0071] Identification of Transformants Containing the
Polynucleotide Sequence
[0072] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, its presence
and expression should be confirmed. For example, if the DBIH
polynucleotide sequence is inserted within a marker gene sequence,
recombinant cells containing DBIH can be identified by the absence
of marker gene function. Alternatively, a marker gene can be placed
in tandem with a DBIH sequence under the control of a single
promoter. Expression of the marker gene in response to induction or
selection usually indicates expression of the tandem DBIH as
well.
[0073] Alternatively, host cells which contain the coding sequence
for DBIH and express DBIH may be identified by a variety of
procedures known to those of skill in the art. These procedures
include, but are not limited to, DNA-DNA or DNA-RNA hybridization
and protein bioassay or immunoassay techniques which include
membrane, solution, or chip based technologies for the detection
and/or quantification of the nucleic acid or protein.
[0074] The presence of the polynucleotide sequence encoding DBIH
can be detected by DNA-DNA or DNA-RNA hybridization or
amplification using probes, portions or fragments of DBIH-encoding
nucleotides. Nucleic acid amplification based assays involve the
use of oligonucleotides or oligomers based on the DBIH sequence to
detect transformants containing DBIH DNA or RNA. As used herein
"oligonucleotides" or "oligomers" refer to a nucleic acid sequence
of at least about 10 nucleotides and as many as about 60
nucleotides, preferably about 15 to 30 nucleotides, and more
preferably about 20-25 nucleotides which can be used as a probe or
amplimer.
[0075] A variety of protocols for detecting and measuring the
expression of DBIH, using either polyclonal or monoclonal
antibodies specific for the protein are known in the art. Examples
include enzyme-linked imimunosorbent assay (ELISA),
radioimmunoassay (RIA) and fluorescent activated cell sorting
(FACS). A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on
DBIH is preferred, but a competitive binding assay may be employed.
These and other assays are described, among other places, in
Hampton, R. et al. (1990, Serological Methods, a Laboratory Manual,
APS Press, St Paul Minn.) and Maddox, D. E. et al. (1983, J. Exp.
Med. 158:1211).
[0076] A wide variety of labels and conjugation techniques are
known by those skilled in the art and can be used in various
nucleic acid and amino acid assays. Means for producing labeled
hybridization or PCR probes for detecting sequences related to DBIH
include oligolabeling, nick translation, end-labeling or PCR
amplification using a labeled nucleotide. Alternatively, the DBIH
sequence, or any portion of it, may be cloned into a vector for the
production of an mRNA probe. Such vectors are known in the art, are
commercially available, and may be used to synthesize RNA probes in
vitro by addition of an appropriate RNA polymerase such as T7, T3
or SP6 and labeled nucleotides.
[0077] A number of companies such as Pharmacia Biotech (Piscataway
N.J.), Promega (Madison Wis.), and US Biochenical Corp (Cleveland
Ohio) supply commercial kits and protocols for these procedures.
Suitable reporter molecules or labels include those radionuclides,
enzymes, fluorescent, cheniluminescent, or chromogenic agents as
well as substrates, cofactors, inhibitors, magnetic particles and
the like. Patents teaching the use of such labels include U.S. Pat.
Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;
4,275,149 and 4,366,241. Also, recombinant immunoglobulins may be
produced as shown in U.S. Pat. No. 4,816,567 incorporated herein by
reference.
[0078] Purification of DBIH
[0079] Host cells transformed with a DBIH-encoding nucleotide
sequence may be cultured under conditions suitable for the
expression and recovery of the encoded protein from cell culture.
The protein produced by a recombinant cell may be contained
intracellularly or secreted depending on the sequence and/or the
vector used. As will be understood by those of skill in the art,
expression vectors containing DBIH can be designed for efficient
production and proper transmembrane insertion of DBIH into a
prokaryotic or eukaryotic cell membrane. Other recombinant
constructions may join DBIH to nucleotide sequence encoding a
polypeptide domain which will facilitate purification of soluble
proteins (Kroll, D. J. et al. (1993) DNA Cell. Biol. 12:441-53; Cf
discussion of vectors infra containing fusion proteins).
[0080] DBIH may also be expressed as a recombinant protein with one
or more additional polypeptide domains added to facilitate protein
purification. Such purification facilitating domains include, but
are not limited to, metal chelating peptides such as
histidine-tryptophan modules that allow purification on immobilized
metals, protein A domains that allow purification on immobilized
immunoglobulin, and the domain utilized in the FLAGS
extension/affinity purification system (Immunex Corp, Seattle
Wash.). The inclusion of a cleavable linker sequence such as Factor
XA or enterokinase (Invitrogen, San Diego Calif.) between the
purification domain and DBIH is useful to facilitate purification.
One such expression vector provides for expression of a fusion
protein compromising an DBIH and contains nucleic acid encoding 6
histidine residues followed by thioredoxin and an enterokinase
cleavage site. The histidine residues facilitate purification on
IMIAC (immobilized metal ion affinity chromatography as described
in Porath et al. (1992) Protein Expression and Purification 3:
263-281) while the enterokinase cleavage site provides a means for
purifying the DBI from the fusion protein.
[0081] In addition to recombinant production, fragments of DBIH may
be produced by direct peptide synthesis using solid-phase
techniques (cf Stewart et al. (1969) Solid-Phase Peptide Synthesis,
WH Freeman Co, San Francisco; Merrifield, J. (1963) J. Am. Chem.
Soc. 85:2149-2154). In vitro protein synthesis maybe performed
using manual techniques orby automation. Automated synthesis may be
achieved, for example, using Applied Biosystems 431A Peptide
Synthesizer (Perkin Ehner, Foster City Calif.) in accordance with
the instructions provided by the manufacturer. Various fragments of
DBIH may be chemically synthesized separately and combined using
chemical methods to produce the full length molecule.
[0082] Uses of DBIH
[0083] The rationale for the use of polynucleotide and polypeptide
sequences disclosed herein is based in part on the chemical and
structural homology among the novel DBIH and the human and bovine
isoforms of DBI/ACBP. DBIH may be used in the diagnosis and
treatment of conditions, disorders or diseases associated with
abnormal function of paraganglia, including paragangliomas.
[0084] The clinical features and morbidity of paragangliomas are
due predominantly to the abnormal release of catecholamines.
Hypertension is the most common manifestation. Paraganglioma is a
correctable cause of high blood pressure. Indeed, it is rarely
fatal if properly diagnosed and treated.
[0085] DBIH may be useful in the regulation of the biosynthesis or
metabolism of biological molecules such as catecholamines in
paraganglia. Molecules associated with paraganglial function, or
their precursors or metabolic products, may bind to DBIH in a
manner analogous to that of fatty acyl-CoAs to DBI/ACBP. DBIH may
sequester and protect the bound ligand from unwanted side-reactions
such as hydrolysis or oxidation, or present the bound ligand to a
receptor molecule. Alternatively, DBIH or a fragment thereof may
act as a neuromodulator of catecholamine-induced responses in
paraganglia.
[0086] DBIH or its fragments can be used to identify specific
molecules which it sequesters or with which it interacts. In this
regard, DBIH may also be used to sequester therapeutic agents,
specifically binding the drug so that the complex is water-soluble
and suitable for therapeutic delivery.
[0087] In overexpression of DBIH associated with
paraganglia-related disorders, it may be advantageous to suppress
DBIH. DBIH could be suppressed by administration of antisense
oligonucleotides. Alternatively, antibodies specifically
recognizing the active site of DBIH may be introduced to treat
diseases or conditions associated with abnormal DBIH activity.
[0088] The structure of DBIH also allows its use in delivery of
other therapeutic molecules. DBIH may bind therapeutic agents or
drugs which are ordinarily insoluble or only slightly soluble in
water. The high charge density of the DBIH molecule renders the
protein-ligand complex soluble in an aqueous environment. The
specificity and binding affinities of DBIH for therapeutic ligands
may be manipulated by protein engineering techniques known to those
skilled in the art.
[0089] DBIH Antibodies
[0090] DBIH-specific antibodies are useful for the diagnosis of
conditions and diseases associated with expression of DBIH. Such
antibodies may include, but are not limited to, polyclonal,
monoclonal, chimeric, single chain, Fab fragments and fragments
produced by a Fab expression library. Neutralizing antibodies,
i.e., those which inhibit dimer formation, are especially preferred
for diagnostics and therapeutics.
[0091] DBIH for antibody induction does not require biological
activity; however, the protein fragment, or oligopeptide must be
antigenic. Peptides used to induce specific antibodies may have an
amino acid sequence consisting of at least five amino acids,
preferably at least 10 amino acids. Preferably, they should mimic a
portion of the amino acid sequence of the natural protein and may
contain the entire amino acid sequence of a small, naturally
occurring molecule. Short stretches of DBIH amino acids may be
fused with those of another protein such as keyhole limpet
hemocyanin and antibody produced against the chimeric molecule.
Procedures well known in the art can be used for the production of
antibodies to DBIH.
[0092] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, etc may be immunized by injection with
DBIH or any portion, fragment or oligopeptide which retains
immunogenic properties. Depending on the host species, various
adjuvants may be used to increase immunological response. Such
adjuvants include but are not limited to, Freund's, mineral gels
such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG
(bacilli Calmette-Guerin) and Corynebacterium parvum are
potentially useful human adjuvants.
[0093] Monoclonal antibodies to DBIH may be prepared using any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include but are not
limited to the hybridoma technique originally described by Koehler
and Milstein (1975) Nature 256:495-497, the human B-cell hybridoma
technique (Kosbor et al. (1983) Immunol. Today 4:72; Cote et al.
(1983) Proc. Natl. Acad. Sci. USA 80:2026-2030) and the
EBV-hybridoma technique (Cole et al. (1985) Monoclonal Antibodies
and Cancer Therapy, Alan R Liss Inc, New York N.Y., pp 77-96).
[0094] In addition, techniques developed for the production of
"chimeric antibodies", the splicing of mouse antibody genes to
human antibody genes to obtain a molecule with appropriate antigen
specificity and biological activity can be used (Morrison et al.
(1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger et al.
(1984) Nature 312:604-608; Takeda et al. (1985) Nature
314:452-454). Alternatively, techniques described for the
production of single chain antibodies (U.S. Pat. No. 4,946,778) can
be adapted to produce DBIH-specific single chain antibodies.
[0095] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening recombinant
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in Orlandi et al. (1989, Proc. Natl. Acad.
Sci. USA 86: 3833-3837), and Winter, G. and Milstein, C. (1991;
Nature 349:293-299).
[0096] Antibody fragments which contain specific binding sites for
DBIH may also be generated. For example, such fragments include,
but are not limited to, the F(ab')2 fragments which can be produced
by pepsin digestion of the antibody molecule and the Fab fragments
which can be generated by reducing the disulfide bridges of the
F(ab')2 fragments. Alternatively, Fab expression hbraries may be
constructed to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity (Huse, W. D. et al.
(1989) Science 256:1275-1281).
[0097] A variety of protocols for competitive binding or
immunoradiometric assays using either polyclonal or monoclonal
antibodies with established specificities are well known in the
art. Such immunoassays typically involve the formation of complexes
between DBIH and its specific antibody and the measurement of
complex formation. A two-site, monoclonal-based immunoassay
utilizing monoclonal antibodies reactive to two noninterfering
epitopes on a specific DBIH protein is preferred, but a competitive
binding assay may also be employed. These assays are described in
Maddox, D. E. et al. (1983, J. Exp. Med. 158:1211).
[0098] Diagnostic Assays using DBIH Specific Antibodies
[0099] Particular DBIH antibodies are useful for the diagnosis of
conditions or diseases characterized by expression of DBIH or in
assays to monitor patients being treated with DBIH, agonists or
inhibitors. Diagnostic assays for DBIH include methods utilizing
the antibody and a label to detect DBIH in human body fluids or
extracts of cells or tissues. The polypeptides and antibodies of
the present invention may be used with or without modification.
Frequently, the polypeptides and antibodies will be labeled by
joining them, either covalently or noncovalently, with a reporter
molecule. A wide variety of reporter molecules are known, several
of which were described above.
[0100] A variety of protocols for measuring DBIH, using either
polyclonal or monoclonal antibodies specific for the respective
protein are known in the art. Examples include enzyme-linked
immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent
activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay utilizing monoclonal antibodies reactive to two
non-interfering epitopes on DBIH is preferred, but a competitive
binding assay may be employed. These assays are described, among
other places, in Maddox, supra.
[0101] In order to provide a basis for diagnosis, normal or
standard values for DBIH expression must be established. This is
accomplished by combining body fluids or cell extracts taken from
normal subjects, either animal or human, with antibody to DBIH
under conditions suitable for complex formation which are well
known in the art. The amount of standard complex formation may be
quantified by comparing various artificial membranes containing
known quantities of DBIH with both control and disease samples from
biopsied tissues. Then, standard values obtained from normal
samples may be compared with values obtained from samples from
subjects potentially affected by disease. Deviation between
standard and subject values establishes the presence of disease
state.
[0102] Drug Screening
[0103] DBIH, its catalytic or immunogenic fragments or
oligopeptides thereof, can be used for screening therapeutic
compounds in any of a variety of drug screening techniques. The
fragment employed in such a test may be free in solution, affixed
to a solid support, borne on a cell surface, or located
intracellularly. The formation of binding complexes, between DBIH
and the agent being tested, may be measured.
[0104] Another technique for drug screening which may be used for
high throughput screening of compounds having suitable binding
affinity to the DBIH is described in detail in "Determination of
Amino Acid Sequence Antigenicity" by Geysen, H. N., WO Application
84103564, published on Sep. 13, 1984, and incorporated herein by
reference. In summary, large numbers of different small peptide
test compounds are synthesized on a solid substrate, such as
plastic pins or some other surface. The peptide test compounds are
reacted with fragments of DBIH and washed. Bound DBIH is then
detected by methods well known in the art. Substantially purified
DBIH can also be coated directly onto plates for use in the
aforementioned drug screening techniques. Alternatively,
non-neutralizing antibodies can be used to capture the peptide and
immobilize it on a solid support.
[0105] This invention also contemplates the use of competitive drug
screening assays in which neutralizing antibodies capable of
binding DBIH specifically compete with a test compound for binding
DBIH. In this manner, the antibodies can be used to detect the
presence of any peptide which shares one or more antigenic
determinants with DBIH.
[0106] Uses of the Polynucleotide Encoding DBIH
[0107] A polynucleotide encoding DBIH, or any part thereof, may be
used for diagnostic and/or therapeutic purposes. For diagnostic
purposes, the DBIH of this invention may be used to detect and
quantitate gene expression in biopsied tissues in which expression
of DBIH may be implicated. The diagnostic assay is useful to
distinguish between absence, presence, and excess expression of
DBIH and to monitor regulation of DBIH levels during therapeutic
intervention. Included in the scope of the invention are
oligonucleotide sequences, antisense RNA and DNA molecules, and
PNAs.
[0108] Another aspect of the subject invention is to provide for
hybridization or PCR probes which are capable of detecting
polynucleotide sequences, including genomic sequences, encoding
DBIH or closely related molecules. The specificity of the probe,
whether it is made from a highly specific region, e.g., 10 unique
nucleotides in the 5' regulatory region, or a less specific region,
eg, especially in the 3' region, and the stringency of the
hybridization or amplification (maximal, high, intermediate or low)
will determine whether the probe identifies only naturally
occurring DBIH, alleles or related sequences.
[0109] Probes may also be used for the detection of related
sequences and should preferably contain at least 50% of the
nucleotides from any of these DBIH encoding sequences. The
hybridization probes of the subject invention may be derived from
the nucleotide sequence of SEQ ID NO:2 or from genomic sequence
including promoter, enhancer elements and introns of the naturally
occurring DBIH. Hybridization probes may be labeled by a variety of
reporter groups, including radionuclides such as .sup.32P or
.sup.35S, or enzymatic labels such as alkaline phosphatase coupled
to the probe via avidin/biotin coupling systems, and the like.
[0110] Other means for producing specific hybridization probes for
DBIH DNAs include the cloning of nucleic acid sequences encoding
DBIH or DBIH derivatives into vectors for the production of mRNA
probes. Such vectors are known in the art and are commercially
available and may be used to synthesize RNA probes in vitro by
means of the addition of the appropriate RNA polymerase as T7 or
SP6 RNA polymerase and the appropriate radioactively labeled
nucleotides.
[0111] Diagnostics
[0112] Polynucleotide sequences encoding DBIH may be used for the
diagnosis of conditions or diseases with which the expression of
DBIH is associated. For example, polynucleotide sequences encoding
DBIH may be used in hybridization or PCR assays of fluids or
tissues from biopsies to detect DBIH expression. The form of such
qualitative or quantitative methods may include Southern or
northern analysis, dot blot or other membrane-based technologies;
PCR technologies; dip stick, pin, chip and ELISA technologies. AU
of these techniques are well known in the art and are the basis of
many commercially available diagnostic kits.
[0113] The DBIH nucleotide sequence disclosed herein provides the
basis for assays that detect activation or induction associated
with inflammation or disease. The DBIH nucleotide sequence may be
labeled by methods known in the art and added to a fluid or tissue
sample from a patient under conditions suitable for the formation
of hybridization complexes. After an incubation period, the sample
is washed with a compatible fluid which optionally contains a dye
(or other label requiring a developer) if the nucleotide has been
labeled with an enzyme. After the compatible fluid is rinsed off,
the dye is quantitated and compared with a standard. If the amount
of dye in the biopsied or extracted sample is significantly
elevated over that of a comparable control sample, the nucleotide
sequence has hybridized with nucleotide sequences in the sample,
and the presence of elevated levels of DBIH nucleotide sequences in
the sample indicates the presence of the associated inflammation
and/or disease.
[0114] Such assays may also be used to evaluate the efficacy of a
particular therapeutic treatment regime in animal studies, in
clinical trials, or in monitoring the treatment of an individual
patient. In order to provide a basis for the diagnosis of disease,
a normal or standard profile for DBIH expression must be
established. This is accomplished by combining body fluids or cell
extracts taken from normal subjects, either animal or human, with
DBIH, or a portion thereof, under conditions suitable for
hybridization or amplification. Standard hybridization may be
quantified by comparing the values obtained for normal subjects
with a dilution series of DBIH run in the same experiment where a
known amount of substantially purified DBIH is used. Standard
values obtained from normal samples may be compared with values
obtained from samples from patients afflicted with DBIH-associated
diseases. Deviation between standard and subject values is used to
establish the presence of disease.
[0115] Once disease is established, a therapeutic agent is
administered and a treatment profile is generated. Such assays may
be repeated on a regular basis to evaluate whether the values in
the profile progress toward or return to the normal or standard
pattern. Successive treatment profiles may be used to show the
efficacy of treatment over a period of several days or several
months.
[0116] Polymerase Chain Reaction (PCR) as described in U.S. Pat.
Nos. 4,683,195 and 4,965,188 provides additional uses for
oligonucleotides based upon the DBIH sequence. Such oligomers are
generally chemically synthesized, but they may be generated
enzymatically or produced from a recombinant source. Oligomers
generally comprise two nucleotide sequences, one with sense
orientation (5'->3') and one with antisense (3'<-5'),
employed under optimized conditions for identification of a
specific gene or condition. The same two oligomers, nested sets of
oligomers, or even a degenerate pool of oligomers may be employed
under less stringent conditions for detection and/or quantitation
of closely related DNA or RNA sequences.
[0117] Additionally, methods which may be used to quantitate the
expression of a particular molecule include radiolabeling (Melby,
P. C. et al. (1993) J. Immunol. Methods 159:235-44) or
biotinylating (Duplaa, C. et al. (1993) Anal. Biochem. 229-36)
nucleotides, coamplification of a control nucleic acid, and
standard curves onto which the experimental results are
interpolated. Quantitation of multiple samples may be speeded up by
running the assay in an ELISA format where the oligomer of interest
is presented in various dilutions and a spectrophotometric or
colorimetric response gives rapid quantitation. A definitive
diagnosis of this type may allow health professionals to begin
aggressive treatment and prevent further worsening of the
condition. Similarly, further assays can be used to monitor the
progress of a patient during treatment. Furthermore, the nucleotide
sequences disclosed herein may be used in molecular biology
techniques that have not yet been developed, provided the new
techniques rely on properties of nucleotide sequences that are
currently known such as the triplet genetic code, specific base
pair interactions, and the like.
[0118] Therapeutic Use
[0119] Based upon its homology to the genes encoding the DBI/ACBPs
and its expression profile, the DBIH polynucleotide disclosed
herein may provide the basis for the design of molecules for the
treatment of diseases associated with abnormal function in
paraganglia.
[0120] Expression vectors derived from retroviruses, adenovirus,
herpes or vaccinia viruses, or from various bacterial plasmids, may
be used for delivery of nucleotide sequences to the targeted organ,
tissue or cell population. Methods which are well known to those
skilled in the art can be used to construct recombinant vectors
which will express antisense DBIH. See, for example, the techniques
described in Sambrook et al. (supra) and Ausubel et al.
(supra).
[0121] The polynucleotides comprising full length cDNA sequence
and/or its regulatory elements enable researchers to use DBIH as an
investigative tool in sense (Youssoufian, H. and H. F. Lodish
(1993) Mol. Cell. Biol. 13:98-104) or antisense (Eguchi et al.
(1991) Annu. Rev. Biochem 60:631-652) regulation of gene function.
Such technology is now well known in the art, and sense or
antisense oligomers, or larger fragments, can be designed from
various locations along the coding or control regions.
[0122] Genes encoding DBIH can be turned off by transfecting a cell
or tissue with expression vectors which express high levels of a
desired DBIH fragment. Such constructs can flood cells with
untranslatable sense or antisense sequences. Even in the absence of
integration into the DNA, such vectors may continue to transcribe
RNA molecules until all copies are disabled by endogenous
nucleases. Transient expression may last for a month or more with a
non-replicating vector (Mettler, I., personal communication) and
even longer if appropriate replication elements are part of the
vector system. As mentioned above, modifications of gene expression
can be obtained by designing antisense molecules, DNA, RNA or PNA,
to the control regions of DBIH, ie, the promoters, enhancers, and
introns. Oligonucleotides derived from the transcription initiation
site, eg, between -10 and +10 regions of the leader sequence, are
preferred. The antisense molecules may also be designed to block
translation of mRNA by preventing the transcript from binding to
ribosomes. Similarly, inhibition canbe achieved using "triple
helix" base-pairing methodology. Triple helix pairing compromises
the ability of the double helix to open sufficiently for the
binding of polymerases, transcription factors, or regulatory
molecules. Recent therapeutic advances using triplex DNA were
reviewed by Gee, J. E. et al. (In: Huber, B. E. and B. I. Carr
(1994) Molecular and Immunologic Approaches, Futura Publishing Co,
Mt. Kisco N.Y.).
[0123] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of RNA. The mechanism of ribozyme action
involves sequence-specific hybridization of the ribozyme molecule
to complementary target RNA, followed by endonucleolytic cleavage.
Within the scope of the invention are engineered hammerhead motif
ribozyme molecules that can specifically and efficiently catalyze
endonucleolytic cleavage of RNA encoding DBIH.
[0124] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites which include the following sequences, GUA,
GUU and GUC. Once identified, short RNA sequences of between 15 and
20 ribonucleotides corresponding to the region of the target gene
containing the cleavage site may be evaluated for secondary
structural features which may render the oligonucleotide
inoperable. The suitability of candidate targets may also be
evaluated by testing accessibility to hybridization with
complementary oligonucleotides using ribonuclease protection
assays.
[0125] Antisense molecules and ribozymes of the invention may be
prepared by any method known in the art for the synthesis of RNA
molecules. These include techniques for chemically synthesizing
oligonucleotides such as solid phase phosphoramidite chemical
synthesis. Alternatively, RNA molecules may be generated by in
vitro and in vivo transcription of DNA sequences encoding DBIH.
Such DNA sequences may be incorporated into a wide variety of
vectors with suitable RNA polymerase promoters such as T7 or SP6.
Alternatively, antisense cDNA constructs that synthesize antisense
RNA constitutively or inducibly can be introduced into cell lines,
cells or tissues.
[0126] RNA molecules may be modified to increase intracellular
stability and half-life. Possible modifications include, but are
not limited to, the addition of flanking sequences at the 5' and/or
3' ends of the molecule or the use of phosphorothioate or 2'
O-methyl rather than phosphodiesterase linkages within the backbone
of the molecule. This concept is inherent in the production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine, queosine and wybutosine as
well as acetyl-, methyl-, thio- and similarly modified forms of
adenine, cytidine, guanine, thymine, and uridine which are not as
easily recognized by endogenous endonucleases.
[0127] Methods for introducing vectors into cells or tissues
include those methods discussed infra and which are equally
suitable for in vivo, in vitro and ex vivo therapy. For ex vivo
therapy, vectors are introduced into stem cells taken from the
patient and clonally propagated for autologous transplant back into
that same patient as presented in U.S. Pat. Nos. 5,399,493 and
5,437,994, disclosed herein by reference. Delivery by transfection
and by liposome are quite well known in the art.
[0128] Furthermore, the nucleotide sequences for DBIH disclosed
herein may be used in molecular biology techniques that have not
yet been developed, provided the new techniques rely on properties
of nucleotide sequences that are currently known, including but not
limited to such properties as the triplet genetic code and specific
base pair interactions.
[0129] Detection and Mapping of Related Polynucleotide
Sequences
[0130] The nucleic acid sequence for DBIH can also be used to
generate hybridization probes for mapping the naturally occurring
genomic sequence. The sequence may be mapped to a particular
chromosome or to a specific region of the chromosome using well
known techniques. These include in situ hybridization to
chromosomal spreads, flow-sorted chromosomal preparations, or
artificial chromosome constructions such as yeast artificial
chromosomes, bacterial artificial chromosomes, bacterial P1
constructions or single chromosome cDNA libraries as reviewed in
Price, C. M. (1993; Blood Rev. 7:127-34) and Trask, B. J. (1991;
Trends Genet. 7:149-54).
[0131] The technique of fluorescent in situ hybridization of
chromosome spreads has been described, among other places, in Verma
et al. (1988) Human Chromosomes: A Manual of Basic Techniques,
Pergamon Press, New York N.Y. Fluorescent in situ hybridization of
chromosomal preparations and other physical chromosome mapping
techniques may be correlated with additional genetic map data.
Examples of genetic map data can be found in the 1994 Genome Issue
of Science (265:1981f). Correlation between the location of a DBIH
on a physical chromosomal map and a specific disease (or
predisposition to a specific disease) may help delimit the region
of DNA associated with that genetic disease. The nucleotide
sequences of the subject invention may be used to detect
differences in gene sequences between normal, carrier or affected
individuals.
[0132] In situ hybridization of chromosomal preparations and
physical mapping techniques such as linkage analysis using
established chromosomal markers may be used for extending genetic
maps. For example, an STS based map of the human genome was
recently published by the Whitehead-MIT Center for Genomic Research
(Hudson, T. J. et al. (1995) Science 270:1945-1954). Often the
placement of a gene on the chromosome of another mammalian species
such as mouse (Whitehead Institute/MIT Center for Genome Research,
Genetic Map of the Mouse, Database Release 10, Apr. 28, 1995) may
reveal associated markers even if the number or arm of a particular
human chromosome is not known. New sequences can be assigned to
chromosomal arms, or parts thereof, by physical mapping. This
provides valuable information to investigators searching for
disease genes using positional cloning or other gene discovery
techniques. Once a disease or syndrome, such as ataxia
telangiectasia (AT), has been crudely localized by genetic linkage
to a particular genomic region, for example, AT to 11q22-23 (Gatti
et al. (1988) Nature 336:577-580), any sequences mapping to that
area may represent associated or regulatory genes for further
investigation. The nucleotide sequence of the subject invention may
also be used to detect differences in the chromosomal location due
to translocation, inversion, etc. among normal, carrier or affected
individuals.
[0133] Pharmaceutical Compositions
[0134] The present invention relates to pharmaceutical compositions
which may comprise nucleotides, proteins, antibodies, agonists,
antagonists, or inhibitors, alone or in combination with at least
one other agent, such as stabilizing compound, which may be
administered in any sterile, biocompatible pharmaceutical carrier,
including, but not limited to, saline, buffered saline, dextrose,
and water. Any of these molecules can be administered to a patient
alone, or in combination with other agents, drugs or hormones, in
pharmaceutical compositions where it is mixed with excipient(s) or
pharmaceutically acceptable carriers. In one embodinent of the
present invention, the pharmaceutically acceptable carrier is
pharmaceutically inert.
[0135] Administration of Pharmaceutical Compositions
[0136] Administration of pharmaceutical compositions is
accomplished orally or parenterally. Methods of parenteral delivery
include topical, intra-arterial (directly to the tumor),
intramuscular, subcutaneous, intramedullary, intrathecal,
intraventricular, intravenous, intraperitoneal, or intranasal
administration. In addition to the active ingredients, these
pharmaceutical compositions may contain suitable pharmaceutically
acceptable carriers comprising excipients and auxiliaries which
facilitate processing of the active compounds into preparations
which can be used pharmaceutically. Further details on techniques
for formulation and administration may be found in the latest
edition of "Remington's Pharmaceutical Sciences" (Maack Publishing
Co, Easton Pa.).
[0137] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral administration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions and the like, for ingestion by the patient.
[0138] Pharmaceutical preparations for oral use can be obtained
through combination of active compounds with solid excipient,
optionally grinding a resulting mixture, and processing the mixture
of granules, after adding suitable auxiliaries, if desired, to
obtain tablets or dragee cores. Suitable excipients are
carbohydrate or protein fillers such as sugars, including lactose,
sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,
potato, or other plants; cellulose such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
and gums including arabic and tragacanth; and proteins such as
gelatin and collagen. If desired, disintegrating or solubilizing
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium
alginate.
[0139] Dragee cores are provided with suitable coatings such as
concentrated sugar solutions, which may also contain gum arabic,
talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol,
and/or titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures. Dyestuffs or pigments may be added to
the tablets or dragee coatings for product identification or to
characterize the quantity of active compound, i.e., dosage.
[0140] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with a
filler or binders such as lactose or starches, lubricants such as
talc or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid paraffin, or liquid
polyethylene glycol with or without stabilizers.
[0141] Pharmaceutical formulations for parenteral administration
include aqueous solutions of active compounds. For injection, the
pharmaceutical compositions of the invention may be formulated in
aqueous solutions, preferably in physiologically compatible buffers
such as Hanks's solution, Ringer's solution, or physiologically
buffered saline. Aqueous injection suspensions may contain
substances which increase the viscosity of the suspension, such as
sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,
suspensions of the active compounds may be prepared as appropriate
oily injection suspensions. Suitable lipophilic solvents or
vehicles include fatty oils such as sesame oil, or synthetic fatty
acid esters, such as ethyl oleate or triglycerides, or liposomes.
Optionally, the suspension may also contain suitable stabilizers or
agents which increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions. For topical or
nasal administration, penetrants appropriate to the particular
barrier to be permeated are used in the formulation. Such
penetrants are generally known in the art.
[0142] Manufacture and Storage
[0143] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is known in the art, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping
or lyophilizing processes.
[0144] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic,
etc. Salts tend to be more soluble in aqueous or other protonic
solvents than are the corresponding free base forms. In other
cases, the preferred preparation may be a lyophilized powder in 1
mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range
of 4.5 to 5.5 that is combined with buffer prior to use.
[0145] After pharmaceutical compositions comprising a compound of
the invention formulated in a acceptable carrier have been
prepared, they can be placed in an appropriate container and
labeled for treatment of an indicated condition. For administration
of DBIH, such labeling would include amount, frequency and method
of administration.
[0146] Therapeutically Effective Dose
[0147] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose.
The determination of an effective dose is well within the
capability of those skilled in the art.
[0148] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays, e.g., of
neoplastic cells, or in animal models, usually mice, rabbits, dogs,
or pigs. The animal model is also used to achieve a desirable
concentration range and route of administration. Such information
can then be used to determine useful doses and routes for
administration in humans.
[0149] A therapeutically effective dose refers to that amount of
protein or its antibodies, antagonists, or inhibitors which
ameliorate the symptoms or condition. Therapeutic efficacy and
toxicity of such compounds can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., ED50 (the dose therapeutically effective in 50% of the
population) and LD50 (the dose lethal to 50% of the population).
The dose ratio between therapeutic and toxic effects is the
therapeutic index, and it can be expressed as the ratio, LD50/ED50.
Pharmaceutical compositions which exhibit large therapeutic indices
are preferred. The data obtained from cell culture assays and
animal studies is used in formulating a range of dosage for human
use. The dosage of such compounds lies preferably within a range of
circulating concentrations that include the ED50 with little or no
toxicity. The dosage varies within this range depending upon the
dosage form employed, sensitivity of the patient, and the route of
administration.
[0150] The exact dosage is chosen by the individual physician in
view of the patient to be treated. Dosage and administration are
adjusted to provide sufficient levels of the active moiety or to
maintain the desired effect. Additional factors which may be taken
into account include the severity of the disease state, eg, tumor
size and location; age, weight and gender of the patient; diet,
time and frequency of administration, drug combination(s), reaction
sensitivities, and tolerance/response to therapy. Long acting
pharmaceutical compositions might be administered every 3 to 4
days, every week, or once every two weeks depending on half-life
and clearance rate of the particular formulation.
[0151] Normal dosage amounts may vary from 0.1 to 100,000
micrograms, up to a total dose of about 1 g, depending upon the
route of administration. Guidance as to particular dosages and
methods of delivery is provided in the literature. See U.S. Pat.
Nos. 4,657,760; 5,206,344; or 5,225,212. Those skilled in the art
will employ different formulations for nucleotides than for
proteins or their inhibitors. Similarly, delivery of
polynucleotides or polypeptides will be specific to particular
cells, conditions, locations, etc.
[0152] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
[0153] I cDNA Library Construction
[0154] The normal tissue used for paraganglion cDNA library
construction was obtained from a 46 year-old male (Lot #0084 ; Mayo
Clinic, Rochester Mich.). The frozen tissue was homogenized and
lysed in guanidinium isothiocyanate solution using a Brinkmann
Homogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury
N.J.). The lysate was centrifuged over a 5.7 M CsCl cushion using
an Beckman SW28 rotor in a Beckman L8-70M Ultracentrifuge (Beckman
Instruments) for 18 hours at 25,000 rpm at ambient temperature. The
RNA was extracted twice with acid phenol pH 4.0 following
Stratagene's RNA isolation protocol and precipitated using 0.3 M
sodium acetate and 2.5 volumes of ethanol, resuspended in
DEPC-treated water and DNase treated for 15 min at 37.degree. C.
The reaction was stopped with an equal volume of acid phenol and
the RNA was isolated using the Qiagen Oligotex kit (QIAGEN Inc,
Chatsworth Calif.) and used to construct the cDNA library.
[0155] The RNA was handled according to the recommended protocols
in the SuperScript Plasmid System for cDNA Synthesis and Plasmid
Cloning (catalog #18248-013; Gibco/BRL), and cDNAs were ligated
into pSport I. The plasmid pSport I was subsequently transformed
into DH5a.TM. competent cells (Cat. #18258-012, Gibco/BRL).
[0156] II Isolation and Sequencing of cDNA Clones
[0157] Plasmid DNA was released from the cells and purified using
the Miniprep Kit (Catalogue # 77468; Advanced Genetic Technologies
Corporation, Gaithersburg Md.). This kit consists of a 96 well
block with reagents for 960 purifications. The recommended protocol
was employed except for the following changes: 1) the 96 wells were
each filled with only 1 ml of sterile Terrific Broth (Catalog #
22711, LIFE TECHNOLOGIES, Gaithersburg Md.) with carbenicillin at
25 mg/L and glycerol at 0.4%; 2) the bacteria were cultured for 24
hours after the wells were inoculated and then lysed with 60 .mu.l
of lysis buffer; 3) a centrifugation step employing the Beckman
GS-6R @2900 rpm for 5 min was performed before the contents of the
block were added to the primary filter plate; and 4) the optional
step of adding isopropanol to TRIS buffer was not routinely
performed. After the last step in the protocol, samples were
transferred to a Beckman 96-well block for storage.
[0158] Alternative methods of purifying plasmid DNA include the use
of MAGIC MINIPREPS DNA Purification System (Catalogue #A7100,
Promega, Madison Wis.)or QIAwell-8 Plasmid, QIAwell PLUS DNA and
QIAwell ULTRA DNA Purification Systems (QIAGEN Chatsworth
Calif.).
[0159] The cDNAs were sequenced by the method of Sanger F and AR
Coulson (1975; J Mol Biol 94:441f), using a Hamilton Micro Lab 2200
(Hamilton, Reno Nev.) in combination with four Peltier Thermal
Cyclers (PTC200 from MJ Research, Watertown Mass.) and Applied
Biosystems 377 or 373 DNA Sequencing Systems (Perkin Elmer), and
the reading frame was determined.
[0160] III Homology Searching of cDNA Clones and Their Deduced
Proteins
[0161] Each cDNA was compared to sequences in GenBank using a
search algorithm developed by Applied Biosystems and incorporated
into the INHERIT-670 Sequence Analysis System. In this algorithm,
Pattern Specification Language (TRW Inc, Los Angeles Calif.) was
used to determine regions of homology. The three parameters that
determine how the sequence comparisons run were window size, window
offset, and error tolerance. Using a combination of these three
parameters, the DNA database was searched for sequences containing
regions of homology to the query sequence, and the appropriate
sequences were scored with an initial value. Subsequently, these
homologous regions were examined using dot matrix homology plots to
distinguish regions of homology from chance matches. Smith-Waterman
alignments were used to display the results of the homology
search.
[0162] Peptide and protein sequence homologies were ascertained
using the INHERIT 670 Sequence Analysis System in a way similar to
that used in DNA sequence homologies. Pattern Specification
Language and parameter windows were used to search protein
databases for sequences containing regions of homology which were
scored with an initial value. Dot-matrix homology plots were
examined to distinguish regions of significant homology from chance
matches.
[0163] BLAST, which stands for Basic Local Alignment Search Tool
(Altschul, S. F. (1993) J. Mol. Evol. 36:290-300; Altschul, S. F.
et al. (1990) J. Mol. Biol. 215:403-10), was used to search for
local sequence alignments. BLAST produces alignments of both
nucleotide and amino acid sequences to determine sequence
similarity. Because of the local nature of the alignments, BLAST is
especially useful in determining exact matches or in identifying
homologs. BLAST is useful for matches which do not contain gaps.
The fundamental unit of BLAST algorithm output is the High-scoring
Segment Pair (HSP).
[0164] An HSP consists of two sequence fragments of arbitrary but
equal lengths whose alignment is locally maximal and for which the
alignment score meets or exceeds a threshold or cutoff score set by
the user. The BLAST approach is to look for HSPs between a query
sequence and a database sequence, to evaluate the statistical
significance of any matches found, and to report only those matches
which satisfy the user-selected threshold of significance. The
parameter E establishes the statistically significant threshold for
reporting database sequence matches. E is interpreted as the upper
bound of the expected frequency of chance occurrence of an HSP (or
set of HSPs) within the context of the entire database search. Any
database sequence whose match satisfies E is reported in the
program output.
[0165] IV Northern Analysis
[0166] Northern analysis is a laboratory technique used to detect
the presence of a transcript of a gene and involves the
hybridization of a labeled nucleotide sequence to a membrane on
which RNAs from a particular cell type or tissue have been bound
(Sambrook et al., supra).
[0167] Analogous computer techniques use BLAST (Altschul, S. F.,
1993 and 1990, supra) to search for identical or related molecules
in nucleotide databases such as GenBank or the LIFESEQ database
(Incyte, Palo Alto Calif.). This analysis is much faster than
multiple, membrane-based hybridizations. In addition, the
sensitivity of the computer search can be modified to determine
whether any particular match is categorized as exact or
homologous.
[0168] The basis of the search is the product score which is
defined as: 1 % sequence identity .times. % maximum BLAST score
100
[0169] and it takes into account both the degree of similarity
between two sequences and the length of the sequence match. For
example, with a product score of 40, the match will be exact within
a 1-2% error; and at 70, the match will be exact. Homologous
molecules are usually identified by selecting those which show
product scores between 15 and 40, although lower scores may
identify related molecules.
[0170] V Extension of DBIH to Full Length or to Recover Regulatory
Elements
[0171] The nucleic acid sequence encoding full length DBIH (SEQ ID
NO:2) is used to design oligonucleotide primers for extending a
partial nucleotide sequence to full length or for obtaining 5'
sequences from genomic libraries. One primer is synthesized to
initiate extension in the antisense direction (XLR) and the other
is synthesized to extend sequence in the sense direction (XLF).
Primers allow the extension of the known DBIH nucleotide sequence
"outward" generating amplicons containing new, unknown nucleotide
sequence for the region of interest. The initial primers are
designed from the cDNA using OLIGO 4.06 Primer Analysis Software
(National Biosciences), or another appropriate program, to be 22-30
nucleotides in length, to have a GC content of 50% or more, and to
anneal to the target sequence at temperatures about
68.degree.-72.degree. C. Any stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations is
avoided.
[0172] The original, selected cDNA libraries, or a human genomic
library are used to extend the sequence; the latter is most useful
to obtain 5' upstream regions. If more extension is necessary or
desired, additional sets of primers are designed to further extend
the known region.
[0173] By following the instructions for the XL-PCR kit (Perkin
Elmer) and thoroughly mixing the enzyme and reaction mix, high
fidelity amplification is obtained. Beginning with 40 pmol of each
primer and the recommended concentrations of all other components
of the kit, PCR is performed using the Peltier Thermal Cycler
(PTC200; MJ Research, Watertown Mass.) and the following
parameters:
1 Step 1 94.degree. C. for 1 min (initial denaturation) Step 2
65.degree. C. for 1 min Step 3 68.degree. C. for 6 min Step 4
94.degree. C. for 15 sec Step 5 65.degree. C. for 1 min Step 6
68.degree. C. for 7 min Step 7 Repeat step 4-6 for 15 additional
cycles Step 8 94.degree. C. for 15 sec Step 9 65.degree. C. for 1
min Step 10 68.degree. C. for 7:15 min Step 11 Repeat steps 8-10
for 12 cycles Step 12 72.degree. C. for 8 min Step 13 4.degree. C.
(and holding)
[0174] A 5-10 .mu.l aliquot of the reaction mixture is analyzed by
electrophoresis on a low concentration (about 0.6-0.8%) agarose
mini-gel to determine which reactions were successful in extending
the sequence. Bands thought to contain the largest products were
selected and cut out of the gel. Further purification involves
using a commercial gel extraction method such as QIAQuick (QIAGEN
Inc). After recovery of the DNA, Klenow enzyme was used to trim
single-stranded, nucleotide overhangs creating blunt ends which
facilitate religation and cloning.
[0175] After ethanol precipitation, the products are redissolved in
13 .mu.l of ligation buffer, 1 .mu.l T4-DNA ligase (15 units) and 1
.mu.l T4 polynucleotide kinase are added, and the mixture is
incubated at room temperature for 2-3 hours or overnight at
16.degree. C. Competent E. coli cells (in 40 .mu.l of appropriate
media) are transformed with 3 .mu.l of ligation mixture and
cultured in 80 .mu.l of SOC medium (Sambrook, J. et al., supra).
After incubation for one hour at 37.degree. C., the whole
transformation mixture is plated on Luria Bertani (LB)-agar
(Sambrook, J. et al., supra) containing 2.times.Carb. The following
day, several colonies are randomly picked from each plate and
cultured in 150 .mu.l of liquid LB/2.times.Carb medium placed in an
individual well of an appropriate, commercially-available, sterile
96-well microtiter plate. The following day, 5 .mu.l of each
overnight culture is transferred into a non-sterile 96-well plate
and after dilution 1:10 with water, 5 .mu.l of each sample is
transferred into a PCR array.
[0176] For PCR amplification, 18 .mu.l of concentrated PCR reaction
mix (3.3.times.) containing 4 units of rTth DNA polymerase, a
vector primer and one or both of the gene specific primers used for
the extension reaction are added to each well. Amplification is
performed using the following conditions:
2 Step 1 94.degree. C. for 60 sec Step 2 94.degree. C. for 20 sec
Step 3 55.degree. C. for 30 sec Step 4 72.degree. C. for 90 sec
Step 5 Repeat steps 2-4 for an additional 29 cycles Step 6
72.degree. C. for 180 sec Step 7 4.degree. C. (and holding)
[0177] Aliquots of the PCR reactions are run on agarose gels
together with molecular weight markers. The sizes of the PCR
products are compared to the original partial cDNAs, and
appropriate clones are selected, ligated into plasmid and
sequenced.
[0178] VI Labeling and Use of Hybridization Probes
[0179] Hybridization probes derived from SEQ ID NO:2 are employed
to screen cDNAs, genomic DNAs or mRNAs. Although the labeling of
oligonucleotides, consisting of about 20 base-pairs, is
specifically described, essentially the same procedure is used with
larger cDNA fragments. Oligonucleotides are designed using
state-of-the-art software such as OLIGO 4.06 (National
Biosciences), labeled by combining 50 pmol of each oligomer and 250
mCi of [.gamma.-.sup.32P] adenosine triphosphate (Amersham, Chicago
Ill.) and T4 polynucleotide kinase (DuPont NEN, Boston Mass.). The
labeled oligonucleotides are substantially purified with Sephadex
G-25 super fine resin column (Pharmacia). A portion containing
10.sup.7 counts per minute of each of the sense and antisense
oligonucleotides is used in a typical membrane based hybridization
analysis of human genomic DNA digested with one of the following
endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II;
DuPont NEN).
[0180] The DNA from each digest is fractionated on a 0.7 percent
agarose gel and transferred to nylon membranes (Nytran Plus,
Schleicher & Schuell, Durham NH). Hybridization is carried out
for 16 hours at 40.degree. C. To remove nonspecific signals, blots
are sequentially washed at room temperature under increasingly
stringent conditions up to 0.1.times.saline sodium citrate and 0.5%
sodium dodecyl sulfate. After XOMAT AR film (Kodak, Rochester N.Y.)
is exposed to the blots in a Phosphoimager cassette (Molecular
Dynamics, Sunnyvale Calif.) for several hours, hybridization
patterns are compared visually.
[0181] VII Antisense Molecules
[0182] The nucleotide sequence encoding DBIH, or any part thereof,
is used to inhibit in vivo or in vitro expression of naturally
occurring DBIH. Although use of antisense oligonucleotides,
comprising about 20 base-pairs, is specifically described,
essentially the same procedure is used with larger cDNA fragments.
An oligonucleotide based on the coding sequence of DBIH as shown in
FIGS. 1A, 1B and 1C is used to inhibit expression of naturally
occurring DBIH. The complementary oligonucleotide is designed from
the most unique 5' sequence as shown in FIGS. 1A, 1B and 1C and
used either to inhibit transcription by preventing promoter binding
to the upstream nontranslated sequence or translation of an DBIH
transcript by preventing the ribosome from binding. Using an
appropriate portion of the leader and 5' sequence of SEQ ID NO:2,
an effective antisense oligonucleotide includes any 15-nucleotides
spanning the region which translates into the signal or early
coding sequence of the polypeptide as shown in FIGS. 1A, 1B and
1C.
[0183] VIII Expression of DBIH
[0184] Expression of DBIH is accomplished by subcloning the cDNAs
into appropriate vectors and transfecting the vectors into host
cells. In this case, the cloning vector, pSport, previously used
for the generation of the cDNA library is used to express DBIH in
E. coli. Upstream of the cloning site, this vector contains a
promoter for .beta.-galactosidase, followed by sequence containing
the amino-terminal Met and the subsequent 7 residues of
.beta.-galactosidase. Immediately following these eight residues is
a bacteriophage promoter useful for transcription and a linker
containing a number of unique restriction sites.
[0185] Induction of an isolated, transfected bacterial strain with
IPTG using standard methods produces a fusion protein which
consists of the first seven residues of .beta.-galactosidase, about
5 to residues of linker, and the full length DBIH. The signal
sequence directs the secretion of DBIH into the bacterial growth
media which can be used directly in the following assay for
activity.
[0186] IX DBIH Activity
[0187] The binding of a ligand to DBIH is assayed by monitoring the
resulting changes in enthalpy (heat production or absorption) in an
isothermal titration microcalorimeter (Micro-Cal Inc, Northampton
Mass.). Titration microcalorimetry measurements do not require
labeling of the ligand or receptor molecules; detection is based
solely on the intrinsic change in the heat of enthalpy upon
binding. Multiple computer-controlled injections of a known volume
of ligand solution are directed into a thermally-controlled chamber
containing DBIH. The change in enthalpy after each injection is
plotted against the number of injections, producing a binding
isotherm The volumes and concentrations of the injected ligand and
of the DBIH solution are used along with the binding isotherm to
calculate values for the number, affinity, and association of DBIH
with the candidate ligand.
[0188] X Production of DBIH Specific Antibodies
[0189] DBIH substantially purified using PAGE electrophoresis
(Sambrook, supra) is used to immunize rabbits and to produce
antibodies using standard protocols. The amino acid sequence
translated from DBIH is analyzed using DNAStar software (DNAStar
Inc) to determine regions of high immunogenicity and a
corresponding oligopolypeptide is synthesized and used to raise
antibodies by means known to those of skill in the art. Analysis to
select appropriate epitopes, such as those near the C-terminus or
in hydrophilic regions (shown in FIG. 3) is described by Ausubel,
F. M. et al. (supra).
[0190] Typically, the oligopeptides are 15 residues in length,
synthesized using an Applied Biosystems Peptide Synthesizer Model
431A using fmoc-chemistry, and coupled to keyhole limpet hemocyanin
(KLH, Sigma) by reaction with
M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel, F. M.
et al., supra). Rabbits are immunized with the oligopeptide-KLH
complex in complete Freund's adjuvant. The resulting antisera are
tested for antipeptide activity, for example, by binding the
peptide to plastic, blockling with 1% BSA, reacting with rabbit
antisera, washing, and reacting with radioiodinated, goat
anti-rabbit IgG.
[0191] XI Purification of Naturally Occurring DBIH Using Specific
Antibodies
[0192] Naturally occurring or recombinant DBIH is substantially
purified by immunoaffinity chromatography using antibodies specific
for DBIH. An immunoaffinity column is constructed by covalently
coupling DBIH antibody to an activated chromatographic resin such
as CnBr-activated Sepharose (Pharmacia Biotech). After the
coupling, the resin is blocked and washed according to the
manufacturer's instructions.
[0193] Cellular fractions from cells containing DBIH are prepared
by solubilization of the whole cell and isolation of subcellular
fractions by differential centrifugation, by the addition of
detergent, or by other methods well known in the art.
Alternatively, soluble DBIH containing a signal sequence may be
secreted in useful quantity into the medium in which the cells are
grown.
[0194] A fractionated DBIH-containing preparation is passed over
the immunoaffinity column, and the column is washed under
conditions that allow the preferential absorbance of DBIH (e.g.,
high ionic strength buffers in the presence of detergent). The
column is eluted under conditions that disrupt antibody/DBIH
binding (eg, a buffer of pH 2-3 or a high concentration of a
chaotrope such as urea or thiocyanate ion), and DBIH is
collected.
[0195] XII Identification of Molecules Which Interact with DBIH
[0196] DBIH is useful as a research tool for identification,
characterization and purification of molecules with which it
interacts. In one embodiment of affinity purification, DBIH is
covalently coupled to a chromatography column. Cells and their
membranes are extracted, endogenous DBIH is removed and various
DBIH-free subcomponents are passed over the column. DBIH-associated
molecules bind to the column by virtue of their biological
affinity. The DBIH-complex is recovered from the column,
dissociated and the recovered molecule is subjected to either
N-terminal protein sequencing or to high-performance liquid
chromatography/mass spectrometry (HPLC/MS). This amino acid
sequence or mass spectral analysis is then used to identify the
captured molecule or, in the case of a protein ligand, to design
degenerate oligonucleotide probes for cloning its gene from an
appropriate cDNA library.
[0197] In an alternate method, monoclonal antibodies are raised
against DBIH and screened to identify those compounds which inhibit
the binding of the antibody to DBIH. These monoclonal antibodies
are then used in affinity purification or expression cloning of
associated molecules.
[0198] Other soluble binding molecules are identified in a similar
manner. DBIH previously labelled with 125I Bolton-Hunter reagent
(Bolton, A. E. and Hunter, W. M. (1973) Biochem. J. 133:529) is
incubated with extracts or biopsied materials derived from cells or
tissues such as paraganglia, rheumatoid synovium, or cerebellum
After incubation, DBIH complexes (which are larger than the size of
the purified DBIH molecule) are identified by a sizing technique
such as size exclusion chromatography or density gradient
centrifugation and are purified by methods known in the art. The
soluble binding protein(s) are subjected to N-terminal sequencing
or mass spectrometry to obtain information sufficient for database
identification, if the soluble protein or molecule is known, or for
cloning, if the soluble protein is unknown.
[0199] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention which are obvious to those skilled in molecular biology
or related fields are intended to be within the scope of the
following claims.
Sequence CWU 1
1
* * * * *