U.S. patent application number 10/119635 was filed with the patent office on 2002-12-19 for methods of determining susceptibility to or presence of schizophrenia, or a disorder related thereto.
Invention is credited to Gogos, Joseph A., Karayiorgou, Maria.
Application Number | 20020193581 10/119635 |
Document ID | / |
Family ID | 26923376 |
Filed Date | 2002-12-19 |
United States Patent
Application |
20020193581 |
Kind Code |
A1 |
Karayiorgou, Maria ; et
al. |
December 19, 2002 |
Methods of determining susceptibility to or presence of
schizophrenia, or a disorder related thereto
Abstract
Variations in the DNA sequence of the human proline
dehydrogenase gene (PRODH) which correlate to an increased
susceptibility to, or presence of schizophrenia or a disease or
disorder related thereto, such as obsessive compulsive disorder
(OCD), bipolar disorder (BP), or major depressive disorder (MDD)
are provided, along with assays to diagnosing schizophrenia or a
disease or disorder related thereto, and evaluating potential drugs
or agents for using in treating such diseases or disorders.
Inventors: |
Karayiorgou, Maria; (New
York, NY) ; Gogos, Joseph A.; (New York, NY) |
Correspondence
Address: |
KLAUBER & JACKSON
411 HACKENSACK AVENUE
HACKENSACK
NJ
07601
|
Family ID: |
26923376 |
Appl. No.: |
10/119635 |
Filed: |
April 10, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10119635 |
Apr 10, 2002 |
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09277262 |
Mar 26, 1999 |
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6395482 |
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09277262 |
Mar 26, 1999 |
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09229530 |
Jan 13, 1999 |
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Current U.S.
Class: |
536/23.1 |
Current CPC
Class: |
C12N 9/0026 20130101;
C12Q 2600/158 20130101 |
Class at
Publication: |
536/23.1 |
International
Class: |
C07H 021/02; C07H
021/04 |
Claims
What is claimed is:
1. An isolated variant allele of a human proline dehydrogenase
(PRODH) gene, wherein said PRODH gene comprises a DNA sequence of
SEQ ID NO:1, and said variant allele comprises a DNA sequence
having at least one variation in SEQ ID NO:1 wherein the at least
one variation comprises: a G to A transition in the third position
of codon 83; a C to T transition in the first position of codon
101; a G to A transition in the second position of codon 101; a C
to T transition in the first position of codon 247; a C to T
transition in the third position of codon 342; a C to T transition
in the third position of codon 421; an A to G transition in the
second position of codon 437; a T to C transition in the first
position of codon 497; or a combination thereof.
2. An isolated nucleic acid molecule hybridizable to an isolated
variant allele of a human PRODH gene of claim 1 under standard
hybridization conditions.
3. The isolated variant allele of claim 1, detectably labeled.
4. The isolated nucleic acid molecule of claim 2, detectably
labeled.
5. The isolated variant allele of claim 3 wherein said detectable
label comprises a radioactive element, a chemical which fluoresces,
or an enzyme.
6. The isolated nucleic acid molecule of claim 4, wherein said
detectable label comprises a radioactive element, a chemical which
fluoresces, or an enzyme.
7. An isolated variant allele of a human PRODH gene, which encodes
a variant proline dehydrogenase comprising an amino acid sequence
comprising at least one variation in SEQ ID NO:2, wherein said at
least one variation comprises: Arg101Trp; Arg101Glu; Glu437Arg; or
a combination thereof.
8. An isolated variant human proline dehydrogenase comprising an
amino acid sequence comprising at least one variation in SEQ ID
NO:2, wherein said at least one variation comprises: Arg101Trp;
Arg101Glu; Glu437Arg; or a combination thereof.
9. An antibody having the variant proline dehydrogenase of claim 8
as an immunogen.
10. The antibody of claim 9, which is a polyclonal antibody.
11. The antibody of claim 9, which is a monoclonal antibody.
12. The antibody of claim 9, which is a chimeric antibody.
13. The antibody of claim 9, detectably labeled.
14. The antibody of claim 13, wherein said detectable label
comprises a radioactive element, a chemical which fluoresces, or an
enzyme.
15. A cloning vector comprising an origin of replication and an
isolated variant allele of a human PRODH gene, wherein said human
PRODH gene comprises a DNA sequence of SEQ ID NO:1, and said
variant allele comprises a DNA sequence having at least one
variation in SEQ ID NO:1, wherein said at least one variation
comprises: a G to A transition in the third position of codon 83; a
C to T transition in the first position of codon 101; a G to A
transition in the second position of codon 101; a C to T transition
in the first position of codon 247; a C to T transition in the
third position of codon 342; a C to T transition in the third
position of codon 421; an A to G transition in the second position
of codon 437; a T to C transition in the first position of codon
497; or a combination thereof.
16. A cloning vector comprising an origin of replication and an
isolated nucleic acid molecule hybridizable under standard
hybridization conditions to an isolated variant allele of a PRODH
gene, wherein said PRODH gene comprises a DNA sequence of SEQ ID
NO:1, and said isolated variant allele comprises a DNA sequence
having at least one variation in SEQ ID NO:1, wherein said at least
one variation comprises: a G to A transition in the third position
of codon 83; a C to T transition in the first position of codon
101; a G to A transition in the second position of codon 101; a C
to T transition in the first position of codon 247; a C to T
transition in the third position of codon 342; a C to T transition
in the third position of codon 421; an A to G transition in the
second position of codon 437; a T to C transition in the first
position of codon 497; or a combination thereof.
17. The cloning vector of either of claim 15 or 16, wherein said
cloning vector is selected from the group consisting of E. coli,
bacteriophages, plasmids, and pUC plasmid derivatives.
18. The cloning vector of claim 17, wherein bacteriophages further
comprise lambda derivatives, plasmids further comprise pBR322
derivatives, and pUC plasmid derivatives further comprise pGEX
vectors, or pmal-c, pFLAG.
19. An expression vector comprising an isolated variant allele of a
human PRODH gene operatively associated with a promoter, wherein
said human PRODH gene comprises a DNA sequence of SEQ ID NO:1, and
said isolated variant allele comprises a DNA sequence comprising at
least one variation in SEQ ID NO:1, wherein said at least one
variation and a variant allele of the present invention comprises a
DNA sequence having at least one variation in SEQ ID NO:1 wherein
the at least one variation comprises: a G to A transition in the
third position of codon 83; a C to T transition in the first
position of codon 101; a G to A transition in the second position
of codon 101; a C to T transition in the first position of codon
247; a C to T transition in the third position of codon 342; a C to
T transition in the third position of codon 421; an A to G
transition in the second position of codon 437; a T to C transition
in the first position of codon 497; or a combination thereof.
20. An expression vector comprising an isolated nucleic acid
molecule operatively associated with a promoter, wherein the
isolated nucleic acid molecule is hybridizable under standard
hybridization conditions to an isolated variant allele of a human
PRODH gene, wherein said human PRODH comprises a DNA sequence of
SEQ ID NO:1, and said variant allele comprises a DNA sequence
having at least one variation in SEQ ID NO:1, wherein said at least
one variation comprises: a G to A transition in the third position
of codon 83; a C to T transition in the first position of codon
101; a G to A transition in the second position of codon 101; a C
to T transition in the first position of codon 247; a C to T
transition in the third position of codon 342; a C to T transition
in the third position of codon 421; an A to G transition in the
second position of codon 437; a T to C transition in the first
position of codon 497; or a combination thereof.
21. The expression vector of either of claim 19 or 20, wherein said
promoter is selected from the group consisting of the immediate
early promoters of hCMV, early promoters of SV40, early promoters
of adenovirus, early promoters of vaccinia, early promoters of
polyoma, late promoters of SV40, late promoters of adenovirus, late
promoters of vaccinia, late promoters of polyoma, the lac the trp
system, the TAC system, the TRC system, the major operator and
promoter regions of phage lambda, control regions of fd coat
protein, 3-phosphoglycerate kinase promoter, acid phosphatase
promoter, and promoters of yeast .alpha. mating factor.
22. A unicellular host transformed or transfected with an
expression vector comprising an isolated variant allele of a human
PRODH gene operatively associated with a promoter, wherein a human
PRODH gene comprises a DNA sequence of SEQ ID NO:1, and said
variant allele comprises a DNA sequence having at least one
variation in SEQ ID NO:1, wherein said at least one variation
comprises: a G to A transition in the third position of codon 83; a
C to T transition in the first position of codon 101; a G to A
transition in the second position of codon 101; a C to T transition
in the first position of codon 247; a C to T transition in the
third position of codon 342; a C to T transition in the third
position of codon 421; an A to G transition in the second position
of codon 437; a T to C transition in the first position of codon
497; or a combination thereof.
23. A unicellular host transformed with an expression vector
comprising an isolated nucleic acid molecule operatively associated
with a promoter, wherein the isolated nucleic acid molecule is
hybridizable under standard hybridization conditions to an isolated
variant allele of a human PRODH gene, wherein said human PRODH gene
comprises a DNA sequence of SEQ ID NO:1, and said variant allele
comprises a DNA sequence having at least one variation in SEQ ID
NO:1, wherein said at least one variation comprises: a G to A
transition in the third position of codon 83; a C to T transition
in the first position of codon 101; a G to A transition in the
second position of codon 101; a C to T transition in the first
position of codon 247; a C to T transition in the third position of
codon 342; a C to T transition in the third position of codon 421;
an A to G transition in the second position of codon 437; a T to C
transition in the first position of codon 497; or a combination
thereof.
24. The unicellular host of either of claim 22 or 23, wherein said
host is selected from the group consisting of E. coli, Pseudonomas,
Bacillus, Strepomyces, yeast, CHO, R1.1, B-W, L-M, COS1, COS7,
BSC1, BSC40, BMT10 and Sf9 cells.
25. A method of producing an a variant human PRODH protein
comprising an amino acid sequence comprising at least one variation
in SEQ ID NO:1, wherein said at least one variation comprises:
Arg101Trp; Arg101Glu; Glu437Arg; or a combination thereof, wherein
said method comprising the steps of: (a) culturing a unicellular
host of either of claim 22 or 23 under conditions that provide for
expression of said variant human PRODH protein; and (b) recovering
said variant human PRODH protein from said unicellular host, said
culture, or both.
26. A method for detecting a susceptibility to, or the presence of,
schizophrenia or a disease or disorder related thereto, wherein the
method comprises measurement of the levels of activity of an enzyme
in a bodily sample, wherein said enzyme is involved in proline
catabolism, and comparison of said levels to a standard, whereby
modulated levels indicate the susceptibility to, or the presence
of, schizophrenia or a disease or disorder related thereto.
27. The method for detecting a susceptibility of claim 26, wherein
a disease or disorder related to schizophrenia comprises obsessive
compulsive disorder (OCD), bipolar disorder (BP) and major
depressive disorder (MDD).
28. The method for detecting a susceptibility as set forth in claim
26, wherein said enzyme is proline dehydrogenase (PRODH) comprising
an amino acid sequence of SEQ ID NO:2, and reduced levels of PRODH
in a bodily sample as compared to a standard indicates the
susceptibility to, or the presence of schizophrenia or a disease or
disorder related thereto.
29. A method for determining a susceptibility in a subject to
schizophrenia, or a disease or disorder related thereto, wherein
the method comprises the steps of: (a) removing a bodily sample
from the subject, wherein the sample comprises a PRODH gene; (b)
determining whether the PRODH gene of the bodily sample comprises a
DNA sequence comprising a variation in SEQ ID NO:1 comprising a T
to C transition in the first position of codon 497, such that the
presence of said at least one variation in said PRODH gene is
indicative of the subject's susceptibility to schizophrenia or a
disease or disorder related thereto, relative to the susecptibility
of a standard.
30. The method of 29, wherein where the variation of PRODH gene is
indicative of an increased susceptibility of the subject to
schizophrenia or a disease or disorder related thereto relative to
the susceptibility of a standard.
31. An assay system for screening drugs and other agents for
ability to treat schizophrenia or a disease or disorder related
thereto, wherein the assay system comprises: (a) culturing an
observable cellular test colony inoculated with a drug or agent;
(b) harvesting a cellular extract from said cellular test colony;
and (c) examining said extract for the presence of PRODH; wherein
an increase or a decrease in a level of activity of said PRODH in
said test colony compared to level of activity of PRODH in the
control indicates the ability of the drug to modulate the
production, stability, degradation or activity of said PRODH.
32. The assay for screening drugs and other agents of claim 31,
wherein an increase in the level or activity of said PRODH in said
test colony compared to a control is indicative of the potential of
the drug to treat schizophrenia or a disease or disorder related
thereto.
33. A test kit to facilitate diagnosis and treatment of
schizophrenia or a disease or disorder related thereto, comprising:
(a) a predetermined amount of a detectably labeled specific binding
partner of a PRODH; (b) other reagents; and (c) directions for use
of said kit.
34. The test kit of claim 33, wherein said labeled immunochemically
reactive component is selected from the group consisting of
polyclonal antibodies to the PRODH, monoclonal antibodies to the
PRODH, fragments thereof, and mixtures thereof.
35. A test kit to facilitate diagnosis and treatment of
schizophrenia or a disease or disorder related thereto in a
eukaryotic cellular sample, comprising: (a) PCR oligonucleotide
primers suitable PRODH detection; (b) other reagents; and (c)
directions for use of said kit.
36. A test kit to facilitate diagnosis and treatment of
schizophrenia or a disease or disorder related thereto in a
subject, wherein the test kit comprises: (a) PCR oligonucleotide
primers suitable for detection of an isolated variant allele of a
PRODH gene in a bodily sample of the subject comprising a DNA
sequence having a variation in SEQ ID NO:1 comprising a T to C
transition in the first position of codon 497; (b) other reagents;
and (c) directions for using said kit.
37. A method of treating schizophrenia or a disease or disorder
related thereto, comprising administering to a mammal a
therapeutically effective amount of a PRODH comprising an amino
acid sequence of SEQ ID NO:2, a conservative variant thereof, a
fragment thereof, or an analog or derivative thereof.
38. The method of claim 37 wherein said PRODH comprising an amino
acid sequence of SEQ ID NO:2 is administered to modulate the course
of therapy where said PRODH is being co-administered with one or
more additional therapeutic agents.
39. A method of determining the schizophrenic-related
pharmacological activity of a compound comprising: administering
the compound to a mammal; determining the level of activity of
PRODH comprising an amino acid sequence of SEQ ID NO:2 in the
mammal; and comparing the level of activity of PRODH in the mammal
to the level of activity of PRODH in a standard to which the
compound was not administered, wherein an increase in the level of
activity of PRODH in the mammal relative the level of activity of
PRODH in the standard is indicative of the ability of the compound
to treat schizophrenia or a disease or disorder related
thereto.
40. An isolated nucleic acid molecule comprising the DNA sequence
of SEQ ID NO:1, degenerate variants thereof, fragments thereof, or
analogs or derivatives thereof.
41. The isolated nucleic acid molecule of claim 40 detectably
labeled.
42. The isolated nucleic acid molecule of claim 41, wherein the
detectable label comprises a radioactive element, a chemical which
fluoresces, or an enzyme.
43. An isolated nucleic acid molecule hybridizable under standard
hybridization conditions to an isolated nucleic acid molecule
comprising a DNA sequence of SEQ ID NO:1, degenerate variants
thereof, fragments thereof, or analogs or derivatives thereof.
44. The isolated nucleic acid molecule of claim 43, detectably
labeled.
45. The isolated nucleic acid molecule of claim 49, wherein the
detectable label comprises a radioactive element, a chemical which
fluoresces, or an enzyme.
46. An isolated nucleic acid molecule which encodes human proline
dehydrogenase protein, wherein the protein comprises an amino acid
sequence of SEQ ID NO:2.
47. The isolated nucleic acid molecule of claim 46, comprising a
DNA sequence of SEQ ID NO:1.
48. An isolated protein comprising an amino acid sequence of SEQ ID
NO:2, conservative variants thereof, fragments thereof, or analogs
or derivatives thereof.
49. An isolated nucleic acid molecule comprising the DNA sequence
of SEQ ID NO:3, degenerate variants thereof, fragments thereof, or
analogs or derivatives thereof.
50. The isolated nucleic acid molecule of claim 49 detectably
labeled.
51. The isolated nucleic acid molecule of claim 50, wherein the
detectable label comprises a radioactive element, a chemical which
fluoresces, or an enzyme.
52. An isolated nucleic acid molecule hybridizable under standard
hybridization conditions to an isolated nucleic acid molecule
comprising a DNA sequence of SEQ ID NO:3, degenerate variants
thereof, fragments thereof, or analogs or derivatives thereof.
53. The isolated nucleic acid molecule of claim 52, detectably
labeled.
54. The isolated nucleic acid molecule of claim 53, wherein the
detectable label comprises a radioactive element, a chemical which
fluoresces, or an enzyme.
55. An isolated nucleic acid molecule which encodes human proline
dehydrogenase protein, wherein the protein comprises an amino acid
sequence of SEQ ID NO:4.
56. The isolated nucleic acid molecule of claim 55, comprising a
DNA sequence of SEQ ID NO:3.
57. An isolated protein comprising an amino acid sequence of SEQ ID
NO:4, conservative variants thereof, fragments thereof, or analogs
or derivatives thereof.
58. A method of identifying drugs or agents useful in treating
schizophrenia or a disease, comprising the steps of: performing an
first pre-pulse inhibition test (PPI) test on an F3 generation
mouse from a cross Pro/Re X C57B1/6J wild-type, wherein the F3
generation mouse has two copies within its genome of an isolated
variant allele of a Prodh gene comprising a DNA sequence of SEQ ID
NO:7 which are capable of expressing a mutant Prodh comprising an
amino acid sequence of SEQ ID NO:8, to obtain a first percentage of
inhibition of startle response; administering the potential drug or
agent to the F3 generation mouse from a cross of Pro/Re X C57B1/6J
wild-type; performing a second PPI test on the F3 generation mouse
from a cross of Pro/Re X C57B1/6J wild-type to obtain a second
percentage of inhibition of startle response; and comparing the
first percentage to the inhibition of startle response with the
second percentage of startle response, wherein an increase in
percentage of inhibition in the second percentage of inhibition
relative to the first percentage of inhibition is indicative of the
ability of the drug or agent to treat schizophrenia or a disease or
disorder related thereto.
59. A method of identifying drugs or agents useful in treating
schizophrenia or a disease, comprising the steps of: a)
administering the drug or agent to an F3 generation mouse from a
cross of Pro/Re X C57B1/6J wild-type, wherein the F3 generation
mouse has two copies within its genome of an isolated variant
allele of a Prodh gene comprising a DNA sequence of SEQ ID NO:7
which are capable of expressing a mutant Prodh comprising an amino
acid sequence of SEQ ID NO:8; b) performing a PPI test on the F3
generation mouse from a cross of Pro/Re X C57B1/6J wild-type
administered the drug or agent to obtain a percentage of inhibition
of the startle response in the mouse; and c) comparing the
percentage of inhibition of the startle response in F3 generation
mouse from a cross of Pro/Re X C57B1/6J wild-type administered the
drug with the percentage of inhibition of the startle response in
an F3 generation mouse from a cross of Pro/Re X C57B1/6J wild-type,
wherein the F3 generation mouse has two copies within its genome of
an isolated Prodh gene comprising a DNA sequence of SEQ ID NO:3
which are capable of expressing a Prodh comprising an amino acid
sequence of SEQ ID NO:4, such that the percentage of inhibition of
the startle response in the medicated mouse is statistically
equivalent to the percentage of inhibition in the mouse capable of
expressing Prodh comprising a DNA sequence of SEQ ID NO:4, then the
drug or agent has the ability to treat schizophrenia or a disease
or disorder related thereto.
60. A method for identifying a drug or agent for use in treating
schizophrenia or a disease or disorder related thereto, comprising
the steps of: a) administering the drug or agent to an F3
generation mouse from a cross of Pro/Re X C57B1/6J wild-type,
wherein the F3 generation mouse has two copies within its genome of
an isolated variant allele of a Prodh gene comprising a DNA
sequence of SEQ ID NO:7 which are capable of expressing a mutant
Prodh comprising an amino acid sequence of SEQ ID NO:8; b)
performing a PPI test on the F3 generation mouse from a cross of
Pro/Re X C57B1/6J wild-type to obtain a percentage of inhibition of
the startle response in the F3 generation mouse from a cross of
Pro/Re X C57B1/6J wild-type which was administered the drug or
agent; and c) comparing the percentage of inhibition of the startle
response in the F3 generation mouse from a cross of Pro/Re X
C57B1/6J wild-type with the percentage of inhibition of the startle
response in an unmedicated F3 generation mouse from a cross of
Pro/Re X C57B1/6J wild-type, wherein the F3 generation mouse has
two copies within its genome of an isolated variant allele of a
Prodh gene comprising a DNA sequence of SEQ ID NO:7 which are
capable of expressing a mutant Prodh comprising an amino acid
sequence of SEQ ID NO:8, wherein an increase in percentage of
inhibition in the percentage of inhibition in the medicated F3
generation mouse relative to the percentage of inhibition in the
unmedicated F3 generation mouse is indicative of the ability of the
drug or agent to treat schizophrenia or a disease or disorder
related thereto.
Description
CROSS REFERENCE TO A RELATED APPLICATION
[0001] This Application is a continuation-in-part of copending U.S.
application Ser. No. 09/229,530 entitled "Methods of Determining
Susceptibility to or presence of schizophrenia, or a disorder
related thereto", which is hereby incorporated by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to isolated nucleic acid
molecules which encode human and murine proline dehydrogenase, and
methods for determining susceptibility to, or the presence of
schizophrenia or a disease or disorder related thereto, such as
obsessive compulsive disorder, bipolar disorder (BP), or major
depressive disorder in a subject by determining levels of proline
dehydrogenase (PRODH) in a bodily sample. Furthermore, the present
invention comprises polymorphisms of the human proline
dehydrogenase (PRODH) gene which correlate to a phenotype closely
related to schizophrenia or a disease or disorder related thereto.
The present invention also relates to various assays for drugs or
agents that can treat schizophrenia or a disease or disorder
related thereto.
BACKGROUND OF THE INVENTION
[0003] It has been posited that the amino acid proline serves as a
modulator of synaptic transmission in the mammalian brain, due to
the selective expression of a brain specific high affinity proline
transporter in a subset of glutamatergic pathways (Fremeau et al.,
1996). Proline transporter is modulated by enckephalins, the
expression of which may be decreased in the brains of patients with
schizophrenia and elevated proline concentration. Furthermore,
recent analysis indicates that endogenous extracellular proline may
regulate the basal function of some glutamate synapses by
maintaining them in a partially potentiated state. Also, elevated
proline concentration has also been previously associated with
behavioral and neurological effects.
[0004] Evidence of an association between schizophrenia
susceptibility and hemizygous deletions in chromosome 22q11 has
been reported. More specifically, three hemizygous cryptic
deletions at 22q11 in a sample of 300 unrelated schizophrenic
patients have been reported and characterized [Karayiorgou et al.,
Proc. Natl. Acad. Sci. U.S.A. 92, 7612 (1995); Karayiorgou et al.,
Amer. J. Med. Genet. 74, 677 (1997)]. The frequency of this
micro-deletion in the general population is estimated to be
approximately 0.02% and no deletions were found in a sample of 200
healthy controls. The identified locus (approximately 1.5 Mb in
size) is located in the proximal part of a region at chromosome
22q11 and has been implicated independently in schizophrenia
susceptibility through linkage studies [Karayiorgou & Gogos,
Neuron 19, 967-979 (1997)]. This locus overlaps with the critical
region involved in the etiology of Velocardio-facial
(VCFS)/DiGeorge (DGS) syndromes [Driscoll et al. 1993].
Furthermore, it has been shown that approximately 29% VCFS children
with 22q11 deletions develop schizophrenia or schizoaffective
disorder in adolescence and adulthood [Pulver et al., 1994], an
estimate confirmed by a more recent independent study [Murphy and
Owen, Am. J. Med. Genet., 74, 660 (1997)]. Deletions in chromosome
22, band q11 (22q11) have been identified among schizophrenia
patients of diverse ethnic origins (Chinese, Israeli, British,
Danish [L. Y. Chow et al., Am. J. Med. Genet. 74, 677 (1997); D.
Gothelf et al., Am. J. Med. Genet. 72, 455 (1997); 0. Mors and H.
Ewald, Am. J. Med. Genet. 74, 677 (1997); Hodginson et al, Am. J.
Med. Genet. 61, 565 (1997)]) and the 22q11 region has been
implicated in early-onset schizophrenia [Yan et al., 1998]. In
addition, the increased rates of comorbid obsessive compulsive
disorder (OCD) or symptoms (OCS) among schizophrenic patients with
the 22q11 microdeletion locus [Karayiorgou et al., 1996, 1997;
Papolos et al., 1996] and similarly increased rates of anxiety, OCS
and OCD in children and adults with the 22q11 microdeletion in the
absence of schizophrenia [Papolos et al., 1996], potentially
indicate that the 22q11 genomic region may harbor one or more genes
predisposing to obsessive compulsive disorder (OCD).
[0005] Moreover, it has been observed that approximately 20% of
schizophrenia patients report obsessions and compulsions, features
that are found in only 1-2% of the general population [Eisen &
Rasmussen 1993; Berman et al., 1995]. Hence, it is possible that
schizophrenia and OCD may share some pathophysiological and genetic
components. One common central processing mechanism that seems to
be affected in patients with schizophrenia and OCD is sensorimotor
gating. Patients with schizophrenia and OCD demonstrate poor
sensorimotor gating of the startle response as measured by impaired
prepulse inhibition of an acoustic response, and this may lead to
sensory overload, distractibility and cognitive fragmentation.
[0006] However, there is no genetic marker available which is
indicative of a subject's susceptibility to schizophrenia, or a
disease related thereto, such as obsessive compulsive disorder
(OCD), bipolar disorder (BP), or major depressive disorder
(MDD).
[0007] Accordingly, what is needed is a genetic marker to assess a
subject's susceptibility to schizophrenia or a disease or disorder
related thereto. Also needed is a genetic marker to diagnose
schizophrenia, and the development of potential drugs or agents
that have applications in treating schizophrenia or a disease or
disorder related thereto, such as OCD, bipolar disorder, or major
depressive disorder.
[0008] The citation of any reference herein should not be construed
as an admission that such reference is available as "Prior Art" to
the instant application.
SUMMARY OF THE INVENTION
[0009] There is provided, in accordance with the present invention,
an isolated nucleic acid molecule which encodes human proline
dehydrogenase, and the amino acid sequence of human proline
dehydrogenase. Also provided is an isolated nucleic acid molecule
comprising a DNA sequence which encodes murine proline
dehydrogenase, and the amino acid sequence of murine proline
dehydrogenase. Furthermore, there is provided, in accordance with
the present invention, methods for determining a subject's
susceptibility to schizophrenia or a disease or disorder related
thereto, such as a schizoaffective disorder or disorders related
thereto, like OCD, bipolar disorder, or major depressive disorder,
using a variant allele of the gene encoding PRODH. Detection of
such a variant allele in the genome of a subject may be indicative
of the subject's susceptibility to schizophrenia. Furthermore, a
variant allele of the PRODH gene can also be used to assay drugs
and agents for potential use in treating schizophrenia or a disease
or disorder related thereto, such as obsessive compulsive disorder
(OCD), bipolar disorder (BP) or major depressive disorder
(MDD).
[0010] Thus broadly, the present invention extends to an isolated
nucleic acid molecule encoding human proline dehydrogenase, wherein
the isolated nucleic acid molecule comprises a DNA sequence of SEQ
ID NO:1, degenerate variants thereof, fragments thereof, or analogs
or derivatives thereof.
[0011] Furthermore, the present invention extends to an isolated
nucleic acid molecule hybridizable under standard hybridization
conditions to the isolated nucleic acid molecule comprising a DNA
sequence of SEQ ID NO:1, degenerate variants thereof, fragments
thereof, or analogs or derivatives thereof.
[0012] The present invention also extends to an isolated nucleic
acid molecule comprising a DNA sequence of SEQ ID NO:1, degenerate
variants thereof, fragments thereof, or analogs or derivatives
thereof, or an isolated nucleic acid molecule hybridzable thereto
under standard hybridization conditions, wherein the nucleic acid
molecule is detectably labeled. Numerous detectable labels have
applications in the present invention. Examples include a
radioactive element, such as the isotopes .sup.3H, .sup.14C,
.sup.32P, .sup.35S, .sup.36Cl, .sup.51Cr, .sup.57Co, .sup.58Co,
.sup.59Fe, .sup.90Y, .sup.251I, .sup.131I, and .sup.186Re, to name
only a few, chemicals which fluoresce, or enzymes such as alkaline
phosphatase or horseradish peroxidase conjugated to an isolated
nucleic acid molecule of the invention.
[0013] Moreover, the present invention extends to an isolated
nucleic acid molecule comprising a DNA sequence of SEQ ID NO:1,
degenerate variants thereof, fragments thereof, or analogs or
derivatives thereof, or an isolated nucleic acid molecule
hybridizable under standard hybridization conditions thereto,
wherein the nucleic acid molecule encodes human PRODH comprising an
amino acid sequence of SEQ ID NO:2, conservative variants thereof,
fragments thereof, or analogs or derivatives thereof.
[0014] Naturally, the present invention extends to an isolated
human proline dehydrogenase protein (PRODH) comprising an amino
acid sequence of SEQ ID NO:2, conservative variants thereof,
fragments thereof, or analogs or derivatives thereof.
[0015] Also, the present invention extends to an antibody having
human proline dehydrogenase comprising an amino acid sequence of
SEQ ID NO:2, conservative variants thereof, fragments thereof, or
analogs or derivatives thereof, as an immunogen. Such an antibody
can be polyclonal, monoclonal, or chimeric. Further, an antibody of
the invention having human PRODH as an immunogen can be detectably
labeled. As explained above, examples of detectable labels having
applications herein include, but certainly are not limited to
radioactive isotopes, such as .sup.3H, .sup.14C, .sup.32P,
.sup.35S, .sup.36Cl, .sup.51Cr, .sup.57Co, .sup.58Co, .sup.59Fe,
.sup.90Y, .sup.125I, .sup.131I, and .sup.186Re, to name only a few.
Chemicals which fluoresce, or enzymes such as alkaline phosphatase
or horseradish peroxidase, can also be used as detectable
labels.
[0016] In addition, the present invention extends to cloning
vectors for creating copies or "cloning" an isolated nucleic acid
molecule of the invention. More specifically, the present invention
extends to a cloning vector comprising an isolated nucleic acid
molecule comprising a DNA sequence of SEQ ID NO:1, degenerate
variants thereof, fragments thereof, or analogs or derivatives
thereof, and an origin of replication. In another embodiment, the
invention extends to a cloning vector comprising an origin of
replication and an isolated nucleic acid molecule hybridizable
under standard hybridization conditions to an isolated nucleic acid
molecule comprising a DNA sequence of SEQ ID NO:1, degenerate
variants thereof, fragments thereof, or analogs or derivatives
thereof.
[0017] Numerous cloning vectors which are commercially available to
the skilled artisan can be used as a cloning vector of the
invention. Further, it is readily within the skill of one of
ordinary skill in the art to insert an isolated nucleic acid
molecule comprising a DNA sequence of SEQ ID NO:1, a degenerate
variant thereof, a fragment thereof, or an analog or derivative
thereof, or an isolated nucleic acid molecule hybridizable thereto
under standard hybridization conditions into a readily available
cloning vector using recombinant DNA techniques to produce a
cloning vector of the invention. In particular, numerous
commercially available cloning vectors have a polylinker site. One
of ordinary skill in the art can readily cut open a cloning vector
at its polylinker using a variety restriction endonucleases, and
then insert the isolated nucleic acid molecule into the vector
using DNA ligase. Furthermore, a skilled artisan can also
manipulate the ends of an isolated nucleic acid molecule of the
invention or fragment thereof to comprise particular restriction
sites, and cut those cites with restriction endonucleases which
also were used to cut open the vector. The restricted isolated
nucleic acid molecule of the invention can then be readily inserted
into a cloning vector of the invention. Any remaining gaps in the
DNA sequence of the vector can then be filled in using individual
deoxynucleotides and DNA ligase. Particular cloning vectors which
have applications in the present invention include, but are not
limited to E. coli, bacteriophages such as lambda derivatives,
plasmids such as pBR322 derivatives, and pUC plasmid derivatives
such as pGEX vectors, or pmal-c, pFLAG, to name only a few.
[0018] Naturally, the present invention extends to an expression
vector for expressing an isolated nucleic acid molecule of the
invention in order to produce human PRODH, conservative variants
thereof, fragments thereof, or analogs or derivatives thereof. In
particular, an expression vector of the invention comprises an
isolated nucleic acid molecule comprising a DNA sequence of SEQ ID
NO:1, degenerate variants thereof, fragments thereof, or analogs or
derivatives thereof, operatively associated with a promoter. In
another embodiment, an expression vector of the invention comprises
an isolated nucleic acid molecule operatively associated with a
promoter, wherein the isolated nucleic acid molecule is hybridzable
under standard hybridization conditions to an isolated nucleic acid
molecule comprising a DNA sequence of SEQ ID NO:1, degenerate
variants thereof, fragments thereof, or analogs or derivatives
thereof.
[0019] Numerous expression vectors can be used to express an
isolated nucleic acid molecule comprising a DNA sequence of SEQ ID
NO:1, degenerate variants thereof, fragments thereof, or analogs or
derivatives thereof, or an isolated nucleic acid molecule
hybridizable thereto under standard hybridization conditions. In
particular, such expression vectors are generally commercially
available to the skilled artisan, and like cloning vectors,
comprise polylinker sites. As a result, commercially available
expression vectors can be manipulated in a fashion similar to to
the manipulation of a cloning vector, which is described above.
Hence a skilled artisan can readily insert an isolated nucleic acid
molecule of the invention into an expression vector such that the
isolated nucleic acid molecule is operatively associated with a
promoter. Examples of expression vectors having applications herein
are described infra.
[0020] Moreover, the present invention extends to a unicellular
host transformed or transfected with an expression vector
comprising an isolated nucleic acid molecule operatively associated
with a promoter, wherein the isolated nucleic acid molecule encodes
a human proline dehydrogenase protein comprising an amino acid
sequence of SEQ ID NO:2, conservative variants thereof, fragments
thereof, or analogs or derivatives thereof. In one embodiment, the
present invention extends to a unicellular host transformed or
transfected with an expression vector comprising an isolated
nucleic acid molecule operatively associated with a promoter,
wherein the isolated nucleic acid molecule comprises a DNA sequence
of SEQ ID NO:1, degenerate variants thereof, fragments thereof, or
analogs or derivatives thereof. In another embodiment, the
invention extends to a unicellular host transformed or transfected
with an expression vector comprising an isolated nucleic acid
molecule operatively associated with a promoter, wherein the
isolated nucleic acid molecule comprises a DNA sequence
hybridizable under standard hybridization conditions to an isolated
nucleic acid molecule comprising a DNA sequence of SEQ ID NO:1, a
degenerate variant thereof, a fragment thereof, or an analog or
derivative thereof. Numerous unicellular hosts which are readily
available to the skilled artisan have applications in the present
invention. Examples include, but certainly are not limited to, E.
coli, Pseudonomas, Bacillus, Strepomyces, yeast, CHO, R1.1, B-W,
L-M, COS1, COS7, BSC1, BSC40, BMT10 and Sf9 cells.
[0021] Naturally, the present invention extends to method for
producing a PRODH comprising an amino acid sequence of SEQ ID NO:2,
conservative variants thereof, fragments thereof, or analogs or
derivatives thereof. In one embodiment, the method comprises the
steps of:
[0022] a) culturing a unicellular host transformed or transfected
with an expression vector comprising an isolated nucleic acid
molecule which comprises a DNA sequence of SEQ ID NO:1, a
degenerate variant thereof, a fragment thereof, or an analog or
derivative thereof, operatively associated with a promoter, under
conditions that provide for expression of the isolated nucleic acid
molecule to produce a protein comprising an amino acid sequence of
SEQ ID NO:2, a conservative variant thereof, a fragment thereof, or
analog or derivative thereof; and
[0023] b) recovering the protein from the unicellular host, the
culture, or both.
[0024] In another embodiment, the method comprises the steps
of:
[0025] a) culturing a unicellular host transformed or transfected
with an expression vector comprising an isolated nucleic acid
molecule operatively associated with a promoter, wherein the
isolated nucleic acid molecule is hybridizable under standard
hybridization conditions to an isolated nucleic acid molecule
comprising a DNA sequence of SEQ ID NO:1, a degenerate variant
thereof, a fragment thereof, or an analog or derivative thereof,
operatively associated with a promoter, under conditions that
provide for expression of the isolated nucleic acid molecule, to
produce a protein comprising an amino acid sequence of SEQ ID NO:2,
a conservative variant thereof, a fragment thereof, or an analog or
derivative thereof; and
[0026] b) recovering the protein from the unicellular host, the
culture, or both.
[0027] In another embodiment, the present invention extends to an
isolated nucleic acid molecule which encodes a human PRODH, wherein
the isolated nucleic acid molecule comprises a DNA sequence of SEQ
ID NO:9, degenerate variants thereof, fragments thereof, or analogs
or derivatives thereof.
[0028] Furthermore, the present invention extends to an isolated
nucleic acid molecule hybridizable under standard hybridization
conditions to the isolated nucleic acid molecule comprising a DNA
sequence of SEQ ID NO:9, degenerate variants thereof, fragments
thereof, or analogs or derivatives thereof.
[0029] The present invention also extends to an isolated nucleic
acid molecule comprising a DNA sequence of SEQ ID NO:9, degenerate
variants thereof, fragments thereof, or analogs or derivatives
thereof, or an isolated nucleic acid molecule hybridzable thereto
under standard hybridization conditions, wherein the nucleic acid
molecule is detectably labeled. Numerous detectable labels have
applications in the present invention and are described infra.
[0030] Moreover, the present invention extends to an isolated
nucleic acid molecule comprising a DNA sequence of SEQ ID NO:9,
degenerate variants thereof, fragments thereof, or analogs or
derivatives thereof, or an isolated nucleic acid molecule
hybridizable under standard hybridization conditions thereto, which
encodes human PRODH comprising an amino acid sequence of SEQ ID
NO:2, conservative variants thereof, fragments thereof, or analogs
or derivatives thereof.
[0031] In addition, the present invention extends to cloning
vectors for creating copies or "cloning" an isolated nucleic acid
molecule of the invention. More specifically, the present invention
extends to a cloning vector comprising an isolated nucleic acid
molecule comprising a DNA sequence of SEQ ID NO:9, degenerate
variants thereof, fragments thereof, or analogs or derivatives
thereof, and an origin of replication. In another embodiment, the
invention extends to a cloning vector comprising an origin of
replication and an isolated nucleic acid molecule hybridizable
under standard hybridization conditions to an isolated nucleic acid
molecule comprising a DNA sequence of SEQ ID NO:9, degenerate
variants thereof, fragments thereof, or analogs or derivatives
thereof.
[0032] Numerous cloning vectors which are commercially available to
the skilled artisan can be used as a cloning vector of the
invention. Further, it is readily within the skill of one of
ordinary skill in the art to insert an isolated nucleic acid
molecule of the present invention or fragment thereof into a
readily available cloning vector using recombinant DNA techniques
to produce a cloning vector of the invention. What's more numerous
cloning vectors having applications herein are readily available to
the skilled artisan, such that with the use of routine experimental
techniques and the new and useful isolated nucleic acid molecule
comprising a DNA sequence of SEQ ID NO:9, one of ordinary skill can
readily produce a cloning vector of the invention. Naturally,
routine recombinant DNA techniques describe here can apply to the
insertion of any isolated nucleic acid molecule of the invention in
to a cloning vector.
[0033] Also, the present invention extends to an expression vector
for expressing an isolated nucleic acid molecule of the invention
in order to produce human PRODH, conservative variants thereof,
fragments thereof, or analogs or derivatives thereof. In
particular, an expression vector of the invention comprises an
isolated nucleic acid molecule comprising a DNA sequence of SEQ ID
NO:9, degenerate variants thereof, fragments thereof, or analogs or
derivatives thereof, operatively associated with a promoter. In
another embodiment, an expression vector of the invention comprises
an isolated nucleic acid molecule operatively associated with a
promoter, wherein the isolated nucleic acid molecule is hybridzable
under standard hybridization conditions to an isolated nucleic acid
molecule comprising a DNA sequence of SEQ ID NO:9, degenerate
variants thereof, fragments thereof, or analogs or derivatives
thereof.
[0034] Numerous expression vectors can be used to express an
isolated nucleic acid molecule comprising a DNA sequence of SEQ ID
NO:9, degenerate variants thereof, fragments thereof, or analogs or
derivatives thereof, or an isolated nucleic acid molecule
hybridizable thereto under standard hybridization conditions. In
particular, such expression vectors are generally commercially
available to the skilled artisan, and like cloning vectors,
comprise polylinker sites. As a result, commercially available
expression vectors can be manipulated in a similar fashion in which
cloning vectors of the invention are manipulated. Hence a skilled
artisan can readily insert an isolated nucleic acid molecule of the
invention into an expression vector such that the isolated nucleic
acid molecule is operatively associated with a promoter. Examples
of expression vectors having applications herein are described
infra.
[0035] Moreover, the present invention extends to a unicellular
host transformed or transfected with an expression vector
comprising an isolated nucleic acid molecule operatively associated
with a promoter, wherein the isolated nucleic acid molecule encodes
a human proline dehydrogenase protein comprising an amino acid
sequence of SEQ ID NO:2, conservative variants thereof, fragments
thereof, or analogs or derivatives thereof. In one embodiment, the
present invention extends to a unicellular host transformed or
transfected with an expression vector comprising an isolated
nucleic acid molecule operatively associated with a promoter,
wherein the isolated nucleic acid molecule comprises a DNA sequence
of SEQ ID NO:9, degenerate variants thereof, fragments thereof, or
analogs of derivatives thereof. In another embodiment, the
invention extends to a unicellular host transformed or transfected
with an expression vector comprising an isolated nucleic acid
molecule operatively associated with a promoter, wherein the
isolated nucleic acid molecule comprises a DNA sequence
hybridizable under standard hybridization conditions to an isolated
nucleic acid molecule comprising a DNA sequence of SEQ ID NO:9, a
degenerate variant thereof, a fragment thereof, or an analog or
derivative thereof. Numerous unicellular hosts which are readily
available to the skilled artisan have applications in the present
invention, and examples are described above.
[0036] Naturally, the present invention extends to method for
producing a PRODH comprising an amino acid sequence of SEQ ID NO:2,
conservative variants thereof, fragments thereof, or analogs or
derivatives thereof. In one embodiment, a method for producing
human proline dehydrogenase comprises the steps of:
[0037] c) culturing a unicellular host of transformed or
transfected with an expression vector comprising an isolated
nucleic acid molecule which comprises a DNA sequence of SEQ ID
NO:9, a degenerate variant thereof, a fragment thereof, or an
analog or derivative thereof, operatively associated with a
promoter, under conditions that provide for expression of the
isolated nucleic acid molecule to produce a protein comprising an
amino acid sequence of SEQ ID NO:2, a conservative variant thereof,
a fragment thereof, or analog or derivative thereof; and
[0038] d) recovering the protein from the unicellular host, the
culture, or both.
[0039] In another embodiment, the method comprises the steps
of:
[0040] a) culturing a unicellular host of transformed or
transfected with an expression vector comprising an isolated
nucleic acid molecule hybridizable under standard hybridization
conditions to an isolated nucleic acid molecule comprising a DNA
sequence of SEQ ID NO:9, a degenerate variant thereof, a fragment
thereof, or an analog or derivative thereof, operatively associated
with a promoter, under conditions that provide for expression of
the isolated nucleic acid molecule, to produce a protein comprising
an amino acid sequence of SEQ ID NO:2, a conservative variant
thereof, a fragment thereof, or an analog or derivative thereof;
and
[0041] b) recovering the protein from the unicellular host, the
culture, or both.
[0042] In another embodiment, the present invention extends to an
isolated nucleic acid molecule which encodes murine proline
dehydrogenase protein (Prodh). In particular, the present invention
extends to an isolated nucleic acid molecule comprising a DNA
sequence of SEQ ID NO:3, degenerate variants thereof, fragments
thereof, or analogs or derivatives thereof. Naturally, the present
invention extends to an isolated nucleic acid molecule which is
hybridizable under standard hybridization conditions to an isolated
nucleic acid molecule comprising a DNA sequence of SEQ ID NO:3,
degenerate variants thereof, fragments thereof, or analogs or
derivatives thereof. As explained above, isolated nucleic acid
molecules of the invention can be detectably labeled. Examples of
detectable labels having applications herein are described
infra.
[0043] In addition, the present invention extends to an isolated
nucleic acid molecule which encodes a Prodh protein comprising an
amino acid sequence of SEQ ID NO:4, conservative variants thereof,
fragments thereof, or analogs or derivatives thereof. A particular
example of such an isolated nucleic acid molecule comprises a DNA
sequence of SEQ ID NO:3, degenerate variants thereof, fragments
thereof, or analogs or derivatives thereof.
[0044] Naturally, the present invention extends to an isolated
Prodh protein comprising an amino acid sequence of SEQ ID NO:4,
conservative variants thereof, fragments thereof, or analogs or
derivatives thereof.
[0045] Further, the present invention extends to an antibody having
a murine proline dehydrogenase protein, a fragment thereof, a
conservative variant thereof, or an analog or derivative thereof as
an immunogen. In a particular example, the immunogen of an antibody
of the invention is a protein comprising an amino acid sequence of
SEQ ID NO:4, conservative variants thereof, fragments thereof, or
analogs or derivatives thereof. Furthermore, such an antibody can
be polyclonal, monoclonal or chimeric, and optionally can be
detectably labeled.
[0046] The present invention further extends to cloning vectors
which can replicate or "clone" an isolated nucleic acid molecule
which encodes a murine proline dehydrogenase protein, conservative
variants thereof, fragments thereof, or analogs or derivatives
thereof. In one embodiment, a cloning vector of the invention
comprises an origin of replication and an isolated nucleic acid
molecule comprising the DNA sequence of SEQ ID NO:3, degenerate
variants thereof, fragments thereof, or analogs or derivatives
thereof. Another embodiment of a cloning vector of the invention
comprises an isolated nucleic acid molecule and an origin of
replication, wherein the isolated nucleic acid molecule is
hybridizable under standard hybridization conditions to an isolated
nucleic acid molecule comprising a DNA sequence of SEQ ID NO:3,
degenerate variants thereof, fragments thereof, or analogs or
derivatives thereof. As explained above, numerous commercially
available cloning vectors which comprise a polylinker region have
ready applications in the present invention. One of ordinary skill
in the art can readily insert an isolated nucleic acid molecule of
the invention into a commercially available cloning vector using
routine recombinant DNA techniques, described infra. Particular
examples of cloning vectors having applications herein include, but
certainly are not limited to E. coli, bacteriophages, plasmids, or
pUC plasmid derivatives. Furthermore, bacteriophage vectors having
applications herein include lambda derivatives, plasmids further
comprise pBR322 derivatives, and pUC plasmid derivatives further
comprise pGEX vectors, or pmal-c, pFLAG, to name only a few.
[0047] In addition, the present invention extends to expression
vectors for producing a murine proline dehydrogenase protein
comprising an amino acid sequence of SEQ ID NO:4, conservative
variants thereof, fragments thereof, or analogs or derivatives
thereof. In particular, the invention extends to an expression
vector comprising an isolated nucleic acid molecule comprising a
DNA sequence of SEQ ID NO:3, degenerate variants thereof, fragments
thereof, or analogs or derivatives thereof, operatively associated
with a promoter. In another embodiment, an expression vector of the
invention comprises an isolated nucleic acid molecule operatively
associated with a promoter, wherein the isolated nucleic acid
molecule is hybridizable under standard hybridizable conditions to
an isolated nucleic acid molecule comprising a DNA sequence of SEQ
ID NO:3, degenerate variants thereof, fragments thereof, or analogs
or derivatives thereof. Numerous expression vectors that are
commercially available have applications herein. Examples of
readily available vectors include derivatives of SV40 and known
bacterial plasmids, e.g., E. coli plasmids col E1, pCR1, pBR322,
pMa1-C2, pET, pGEX (Smith et al., 1988, Gene 67:31-40), pMB9 and
their derivatives, plasmids such as RP4; phage DNAS, e.g., the
numerous derivatives of phage .lambda., e.g., NM989, and other
phage DNA, e.g., M13 and filamentous single stranded phage DNA;
yeast plasmids such as the 2.mu. plasmid or derivatives thereof;
vectors useful in eukaryotic cells, such as vectors useful in
insect or mammalian cells; vectors derived from combinations of
plasmids and phage DNAs, such as plasmids that have been modified
to employ phage DNA or other expression control sequences; and the
like.
[0048] Insertion of an isolated nucleic acid molecule into such an
expression vector is readily within the skill of one of ordinary
skill in the art using recombinant DNA techniques described
herein.
[0049] Further, numerous promoters have applications herein.
Examples of such promoters include, but certainly are not limited
to immediate early promoters of hCMV, early promoters of SV40,
early promoters of adenovirus, early promoters of vaccinia, early
promoters of polyoma, late promoters of SV40, late promoters of
adenovirus, late promoters of vaccinia, late promoters of polyoma,
the lac system the trp system, the TAC system, the TRC system, the
major operator and promoter regions of phage lambda, control
regions of fd coat protein, 3-phosphoglycerate kinase promoter,
acid phosphatase promoter, or promoters of yeast .alpha. mating
factor, to name only a few.
[0050] The present invention further extends to unicellular hosts
transformed or transfected with an expression vector of the
invention. In particular, the present invention extends to a
unicellular host transformed or transfected with an expression
vector comprising an isolated nucleic acid molecule comprising a
DNA sequence of SEQ ID NO:3, degenerate variants thereof, fragments
thereof, or analogs or derivatives thereof, operatively associated
with a promoter. In another embodiment, the present invention
extends to a unicellular host transformed or transfected with an
expression vector comprising an isolated nucleic acid molecule
operatively associated with a promoter, wherein the isolated
nucleic acid molecule is hybridizable under standard hybridization
conditions to an isolated nucleic acid molecule comprising a DNA
sequence of SEQ ID NO:3, degenerate variants thereof, fragments
thereof, or analogs or derivative thereof. Examples of unicellular
hosts having applications herein include but certainly are not
limited to E. coli, Pseudonomas, Bacillus, Strepomyces, yeast, CHO,
R1.1, B-W, L-M, COS1, COS7, BSC1, BSC40, BMT10 or Sf9 cells.
[0051] Naturally, the present invention extends to methods of
producing a murine proline dehydrogenase protein comprising an
amino acid sequence of SEQ ID NO:4, conservative variants thereof,
fragments thereof, or analogs or derivatives thereof. In one
embodiment, the method comprises the steps of:
[0052] a) culturing a unicellular host transformed or transfected
with an expression vector comprising an isolated nucleic acid
molecule comprising a DNA sequence of SEQ ID NO:3, a degenerate
variant thereof, a fragment thereof, or an analog or derivative
thereof, operatively associated with a promoter, under conditions
that provide for expression of the isolated nucleic acid molecule
to produce a protein comprising an amino acid sequence of SEQ ID
NO:4, conservative variant thereof, fragment thereof, or analog or
derivative thereof; and
[0053] b) recovering the protein from the unicellular host, the
culture, or both.
[0054] In another embodiment, the method comprises the steps
of:
[0055] a) culturing a unicellular host transformed or transfected
with an expression vector comprising an isolated nucleic acid
molecule operatively associated with a promoter, wherein the
isolated nucleic acid molecule is hybridizable under standard
hybridization conditions to an isolated nucleic acid molecule
comprising a DNA sequence of SEQ ID NO:3, degenerate variants
thereof, fragments thereof, or analogs or derivatives thereof under
conditions that provide for expression of the isolated nucleic acid
molecule to produce a protein comprising an amino acid sequence of
SEQ ID NO:4, conservative variant thereof, fragment thereof, or
analog or derivative thereof; and
[0056] b) recovering the protein from the unicellular host, the
culture, or both.
[0057] The present invention further extends to an isolated variant
allele of a human proline dehydrogenase (PRODH) gene, wherein the
PRODH gene comprises a DNA sequence of SEQ ID NO:1, and the variant
allele comprises a DNA sequence having at least one variation in
SEQ ID NO:1, wherein the at least one variation comprises:
[0058] a G to A transition in the third position of codon 83;
[0059] a C to T transition in the first position of codon 101;
[0060] a G to A transition in the second position of codon 101;
[0061] a C to T transition in the first position of codon 247;
[0062] a C to T transition in the third position of codon 342;
[0063] a C to T transition in the third position of codon 421;
[0064] an A to G transition in the second position of codon
437;
[0065] a T to C transition in the first position of codon 497;
or
[0066] a combination thereof.
[0067] Moreover, the present invention extends to an isolated
nucleic acid molecule hybridizable under standard hybridization
conditions to an isolated variant allele of a human PRODH gene,
wherein the isolated variant allele comprises a DNA sequence having
at least one variation in SEQ ID NO:1, and the at least one
variation comprises:
[0068] a G to A transition in the third position of codon 83;
[0069] a C to T transition in the first position of codon 101;
[0070] a G to A transition in the second position of codon 101;
[0071] a C to T transition in the first position of codon 247;
[0072] a C to T transition in the third position of codon 342;
[0073] a C to T transition in the third position of codon 421;
[0074] an A to G transition in the second position of codon
437;
[0075] a T to C transition in the first position of codon 497;
or
[0076] a combination thereof.
[0077] In addition, the present invention extends to a detectably
labeled isolated variant allele of a PRODH gene, wherein the PRODH
gene comprises a DNA sequence of SEQ ID NO:1, and the variant
allele comprises a DNA sequence having at least one variation in
SEQ ID NO:1, wherein the at least one variation comprises:
[0078] a G to A transition in the third position of codon 83;
[0079] a C to T transition in the first position of codon 101;
[0080] a G to A transition in the second position of codon 101;
[0081] a C to T transition in the first position of codon 247;
[0082] a C to T transition in the third position of codon 342;
[0083] a C to T transition in the third position of codon 421;
[0084] an A to G transition in the second position of codon
437;
[0085] a T to C transition in the first position of codon 497;
or
[0086] a combination thereof.
[0087] Numerous detectable labels have applications in the present
invention. For example the detectable label can be a radioactive
element, such as the isotopes .sup.3H, .sup.14C, .sup.32P,
.sup.35S, .sup.36Cl, .sup.51Cr, .sup.57Co, .sup.58Co, .sup.59Fe,
.sup.90Y, .sup.125I, .sup.131I, and .sup.186Re, to name only a few.
Chemicals which fluoresce, or enzymes such as alkaline phosphatase
or horseradish peroxidase, can also be used as detectable
labels.
[0088] Moreover, the present invention extends to a detectably
labeled isolated nucleic acid molecule hybridizable under standard
hybridization conditions to an isolated variant allele of a PRODH
gene, wherein the PRODH gene comprises a DNA sequence of SEQ ID
NO:1, and the variant allele comprises a DNA sequence having at
least one variation in SEQ ID NO:1, wherein the at least one
variation comprises:
[0089] a G to A transition in the third position of codon 83;
[0090] a C to T transition in the first position of codon 101;
[0091] a G to A transition in the second position of codon 101;
[0092] a C to T transition in the first position of codon 247;
[0093] a C to T transition in the third position of codon 342;
[0094] a C to T transition in the third position of codon 421;
[0095] an A to G transition in the second position of codon
437;
[0096] a T to C transition in the first position of codon 497;
or
[0097] a combination thereof.
[0098] Detectable labels set forth throughout the specification
have applications in such an isolated nucleic acid molecule.
[0099] In addition, the present invention extends to an isolated
variant allele of a PRODH gene which encodes a variant human
proline PRODH comprising at least one variation in the amino acid
sequence of PRODH, wherein PRODH comprises an amino acid sequence
of SEQ ID NO:2, and is encoded by a human proline dehydrogenase
gene comprising a DNA sequence of SEQ ID NO:1. A variant PRODH
protein of the present invention comprises an amino acid sequence
having at least one variation in SEQ ID NO:2, wherein the at least
one variation comprises:
[0100] Arg101Trp;
[0101] Arg101Glu;
[0102] Glu437Arg; or
[0103] a combination thereof.
[0104] In particular, a variant allele of a human PRODH gene
comprising a DNA sequence comprising a C to T transition in the
first position of codon 101 of SEQ ID NO:1, encodes a variant human
PRODH protein comprising an amino acid sequence comprising an
Arg101Trp variation in SEQ ID NO:2. Furthermore, a variant allele
of a human PRODH gene comprising a DNA sequence having a G to A
transition in the second position of codon 101 of SEQ ID NO:1
encodes a variant human PRODH protein comprising an amino acid
sequence having an Arg101Glu variation in SEQ ID NO:2. Hence
naturally, a variant allele of a human PRODH gene comprising a DNA
sequence having an A to G transition in the second position of
codon 437 of SEQ ID NO:1 encodes a variant human PRODH protein
comprising an amino acid sequence having a Glu437Arg variation in
SEQ ID NO:2. As explained above, the present invention also extends
to a variant allele of a human PRODH gene comprising a combination
of variations set forth herein, which encode a variant human PRODH
protein comprising an amino acid sequence having a combination of
amino acid residue variations in SEQ ID NO:2 as described
above.
[0105] Naturally, the present invention extends to an isolated
variant human PRODH protein comprising an amino acid sequence
having at least one variation in SEQ ID NO:2, wherein the at least
one variation comprises:
[0106] Arg101Trp;
[0107] Arg101Glu;
[0108] Glu437Arg; or
[0109] a combination thereof.
[0110] Furthermore, the present invention extends to an antibody
having a variant proline dehydrogenase protein of the present
invention as an immunogen. Such an antibody can be a polyclonal
antibody, a monoclonal antibody, or a chimeric antibody. Moreover,
an antibody of the present invention can be detectably labeled.
Examples of detectable labels which have applications in this
embodiment comprises a radioactive element, a chemical which
fluoresces, or an enzyme, to name only a few.
[0111] In addition, the present invention extends to cloning
vectors that can be used to clone copies of a variant allele of a
PRODH gene of the present invention. An example of such a cloning
vector comprises an origin of replication and an isolated variant
allele of a human PRODH gene, wherein the PRODH gene comprises a
DNA sequence of SEQ ID NO:1, and an isolated variant allele of the
PRODH gene comprises a DNA sequence having at least one variation
in SEQ ID NO:1 wherein the at least one variation comprises:
[0112] a G to A transition in the third position of codon 83;
[0113] a C to T transition in the first position of codon 101;
[0114] a G to A transition in the second position of codon 101;
[0115] a C to T transition in the first position of codon 247;
[0116] a C to T transition in the third position of codon 342;
[0117] a C to T transition in the third position of codon 421;
[0118] an A to G transition in the second position of codon
437;
[0119] a T to C transition in the first position of codon 497;
or
[0120] a combination thereof.
[0121] Moreover, the present invention extends to a cloning vector
comprising an origin of replication and an isolated nucleic acid
molecule hybridizable under standard hybridization conditions to an
isolated variant allele of a PRODH gene, wherein the PRODH gene
comprises a DNA sequence of SEQ ID NO:1, and the isolated variant
allele of the PRODH gene comprises a DNA sequence having at least
one variation in SEQ ID NO:1 wherein the at least one variation
comprises:
[0122] a G to A transition in the third position of codon 83;
[0123] a C to T transition in the first position of codon 101;
[0124] a G to A transition in the second position of codon 101;
[0125] a C to T transition in the first position of codon 247;
[0126] a C to T transition in the third position of codon 342;
[0127] a C to T transition in the third position of codon 421;
[0128] an A to G transition in the second position of codon
437;
[0129] a T to C transition in the first position of codon 497;
or
[0130] a combination thereof.
[0131] Numerous cloning vectors have applications in the present
invention and are readily available to a skilled artisan.
Furthermore, it is well within the knowledge of one of ordinary
skill in the art to insert an isolated PRODH variant allele of the
present invention into a commercially available cloning vector
using recombinant DNA techniques described infra. Examples of a
cloning vector having applications in the present invention include
E. coli, bacteriophages, such as lambda derivatives, plasmids, such
as pBR322 derivatives, and pUC plasmid derivatives, such as pGEX
vectors, or pmal-c, pFLAG, to name only a few.
[0132] The present invention further extends to a unicellular host
transformed or transfected with a cloning vector which comprises an
isolated variant allele of the human PRODH gene as described above.
Examples of hosts which are readily available to the skilled
artisan and can be transformed or transfected with a cloning vector
of the present invention include, but are not limited to E. coli,
Pseudonomas, Bacillus, Strepomyces, yeast, CHO, R1.1, B-W, L-M,
COS1, COS7, BSC1, BSC40, BMT10 or Sf9 cells.
[0133] The present invention further extends to a method of cloning
or producing "copies" of an isolated variant allele of a human
PRODH gene of the invention, which comprises inserting a cloning
vector into a unicellular host, and then inducing the host to self
replicate. During the self replication of the host, the origin of
replication of the cloning vector causes the replication of the
cloning vector. After the unicellular host has self replicated
numerous times, the cloning vectors can be isolated from the cloned
host, and the isolated nucleic acid molecule can be isolated via
restriction digestion from the cloning vectors.
[0134] Naturally, the present invention extends to expression
vectors comprising an isolated variant allele of a PRODH gene
operatively associated with a promoter, wherein the PRODH gene
comprises a DNA sequence of SEQ ID NO:1, and an isolated variant
allele of the present invention comprises a DNA sequence having at
least one variation in SEQ ID NO:1 wherein the at least one
variation comprises:
[0135] a G to A transition in the third position of codon 83;
[0136] a C to T transition in the first position of codon 101;
[0137] a G to A transition in the second position of codon 101;
[0138] a C to T transition in the first position of codon 247;
[0139] a C to T transition in the third position of codon 342;
[0140] a C to T transition in the third position of codon 421;
[0141] an A to G transition in the second position of codon
437;
[0142] a T to C transition in the first position of codon 497;
or
[0143] a combination thereof.
[0144] Furthermore, the present invention extends to an expression
vector comprising an isolated nucleic acid molecule operatively
associated with a promoter, wherein the isolated nucleic acid
molecule is hybridizable under standard hybridization conditions to
an isolated variant allele of a PRODH gene, wherein the PRODH gene
comprises a DNA sequence of SEQ ID NO:1, and an isolated variant
allele of the present invention comprises a DNA sequence having at
least one variation in SEQ ID NO:1 wherein the at least one
variation comprises:
[0145] a G to A transition in the third position of codon 83;
[0146] a C to T transition in the first position of codon 101;
[0147] a G to A transition in the second position of codon 101;
[0148] a C to T transition in the first position of codon 247;
[0149] a C to T transition in the third position of codon 342;
[0150] a C to T transition in the third position of codon 421;
[0151] an A to G transition in the second position of codon
437;
[0152] a T to C transition in the first position of codon 497;
or
[0153] a combination thereof.
[0154] Numerous promoters which are readily available to a skilled
artisan, have applications in any expression vector of the
invention. For example, immediate early promoters of hCMV, early
promoters of SV40, early promoters of adenovirus, early promoters
of vaccinia, early promoters of polyoma, late promoters of SV40,
late promoters of adenovirus, late promoters of vaccinia, late
promoters of polyoma, the lac system, the trp system, the TAC
system, the TRC system, the major operator and promoter regions of
phage lambda, control regions of fd coat protein,
3-phosphoglycerate kinase promoter, acid phosphatase promoter, or
promoters of yeast .alpha. mating factor, to name only a few, have
applications herein. Furthermore, it is well within the knowledge
of one of ordinary skill in the art to insert an isolated variant
allele of the invention, or an isolated nucleic acid molecule
hybridizable under standard hybridization conditions to an isolated
variant allele of the invention into a commercially available
expression vector, using recombinant DNA techniques described
infra.
[0155] In addition, the present invention extends to a unicellular
host transformed or transfected with an expression vector of the
present invention. Examples of hosts which can be transformed or
transfected with an expression vector of the present invention, and
have applications in the present invention, include, but are not
limited to, E. coli, Pseudonomas, Bacillus, Strepomyces, yeast,
CHO, R1.1, B-W, L-M, COS1, COS7, BSC1, BSC40, BMT10or Sf9
cells.
[0156] Naturally, the present invention extends to a method for
producing a variant human PRODH protein comprising an amino acid
having at least one variation in SEQ ID NO:2, wherein the at least
one variation comprises
[0157] Arg101Trp;
[0158] Arg101Glu;
[0159] Glu437Arg; or
[0160] a combination thereof.
[0161] An example of such a method comprises the steps of culturing
a unicellular host transformed or transfected with an expression
vector comprising an isolated variant allele of a PRODH gene
operatively associated with a promoter, wherein the isolated
variant allele of the PRODH gene comprises a DNA sequence having at
least one variation in SEQ ID NO:1, and the at least one variation
comprises:
[0162] a G to A transition in the third position of codon 83;
[0163] a C to T transition in the first position of codon 101;
[0164] a G to A transition in the second position of codon 101;
[0165] a C to T transition in the first position of codon 247;
[0166] a C to T transition in the third position of codon 342;
[0167] a C to T transition in the third position of codon 421;
[0168] an A to G transition in the second position of codon
437;
[0169] a T to C transition in the first position of codon 497;
or
[0170] a combination thereof,
[0171] under conditions that provide for expression of the variant
allele. The variant PRODH protein produced from such expression is
then recovered from the unicellular host, the culture, or both.
[0172] Yet another method of the present invention for producing a
variant PRODH protein involves culturing a unicellular host
transformed or transfected with an expression vector comprising an
isolated nucleic acid molecule operatively associated with a
promoter, wherein the isolated nucleic acid molecule is
hybridizable under standard hybridization conditions to an isolated
variant allele of a PRODH gene comprising a DNA sequence having at
least one variation in SEQ ID NO:1, wherein the at least one
variation comprises:
[0173] a G to A transition in the third position of codon 83;
[0174] a C to T transition in the first position of codon 101;
[0175] a G to A transition in the second position of codon 101;
[0176] a C to T transition in the first position of codon 247;
[0177] a C to T transition in the third position of codon 342;
[0178] a C to T transition in the third position of codon 421;
[0179] an A to G transition in the second position of codon
437;
[0180] a T to C transition in the first position of codon 497;
or
[0181] a combination thereof,
[0182] under conditions that provide for expression of the isolated
nucleic acid molecule. The variant human PRODH protein produced
from such induced expression is then recovered from the unicellular
host, the culture, or both.
[0183] Furthermore, the present invention extends to a method for
detecting a susceptibility to, or the presence of schizophrenia or
a disease or disorder related thereto, such as obsessive compulsive
disorder (OCD), bipolar disorder (BP) or major depressive disorder
in a subject, wherein the method comprises measurement of the
levels of activity of an enzyme in a bodily sample which is
involved in proline catabolism. A comparison of the measurement of
the levels of activity of the enzyme in the bodily sample is then
made with the levels of activity of the enzyme in a standard. A
modulated level of enzyme activity in the sample relative to the
level of activity in the standard is indicative of a susceptibility
to, or the presence of, schizophrenia or a disease or disorder
related thereto, such as obsessive compulsive disorder (OCD),
bipolar disorder (BP) or major depressive disorder, in the subject.
In a particular embodiment, the enzyme involved in proline
catabolism is proline dehydrogenase (PRODH), and a reduced level of
activity of PRODH in a bodily sample from the subject compared to
the level of PRODH activity in the standard is indicative of
increased susceptibility to, or the presence of schizophrenia or a
disease or disorder related thereto in the subject relative to the
susceptibility of the standard. Methods of assaying activity of
proline dehydrogenase in a bodily sample are readily available to
the skilled artisan.
[0184] The present invention further extends to a method for
determining a susceptibility to, or the presence of schizophrenia
or a disease or disorder related thereto in a subject, such as
obsessive compulsive disorder (OCD), bipolar disorder (BP) or major
depressive disorder (MDD), wherein the method comprises the steps
of:
[0185] a) removing a bodily sample from the subject, wherein the
sample comprises a PRODH gene; and
[0186] b) determining whether the PRODH gene of the bodily sample
comprises a DNA sequence having a variation in SEQ ID NO:1
comprising a T to C transition in the first position of codon 497.
The presence of the variant allele the PRODH gene in a bodily
sample of the subject indicates the subject has an increased
susceptibility to schizophrenia or a disease or disorder related
thereto, such as obsessive compulsive disorder (OCD), bipolar
disorder (BP) or major depressive disorder (MDD) relative to the
susceptibility of a standard, wherein the bodily sample of the
standard comprises a PRODH gene comprising a DNA sequence of SEQ ID
NO:1.
[0187] Furthermore, the present invention extends to an assay for
screening drugs and other agents for ability to treat schizophrenia
or a disease or disorder related thereto. Such an assay of the
present invention comprises the steps of culturing an observable
cellular test colony which produces PRODH and which has been
inoculated with the drug or agent to be assayed, harvesting a
cellular extract from the cellular test colony, and determining the
level of activity of PRODH in the test colony. An increase or
decrease in the level of activity of PRODH in this test colony
compared to a control test colony not inoculated with the drug, or
compared to the level of activity of PRODH in the cellular test
colony prior to inoculation with the drug or agent, is indicative
of the ability of the drug or agent to modulate the production,
stability, degradation or activity of PRODH, which in turn is
indicative of the drug or agent's ability to treat schizophrenia or
a disease or disorder related thereto, such as obsessive compulsive
disorder (OCD), bipolar disorder (BP) or major depressive disorder.
An increase in the level of activity of PRODH in the test colony
after inoculation with the drug or agent compared to the level of
activity in the control colony, or in the cellular test colony
prior to inoculation with the drug or agent, indicates the drug has
the ability to be used to treat schizophrenia or a disorder related
thereto.
[0188] In another embodiment, the present invention extends to an
assay system which may be prepared in the form of a test kit for
the quantitative analysis of the extent of the presence and/or
activity of PRODH, or to identify drugs or other agents that may
potentiate or increase such activity. Broadly, a system or test kit
of the present invention may comprise a labeled component prepared
by one of the radioactive and/or enzymatic techniques discussed
herein, coupling the label to PRODH, its agonists and/or
antagonists, and one or more additional immunochemical reagents, at
least one of which is a free or immobilized ligand, capable either
of binding with the labeled component, its binding partner, one of
the components to be determined, or their binding partner(s). The
system or test kit may also comprise a polymerase chain reaction
based (PCR) assay which can be used to quantify the PRODH levels of
a sample.
[0189] Hence, the present invention extends to a test kit to
facilitate diagnosis and treatment of schizophrenia or a disease or
disorder related thereto, such as obsessive compulsive disorder
(OCD), bipolar disorder (BP) or major depressive disorder,
comprising:
[0190] (a) a predetermined amount of a detectably labeled specific
binding partner of a PRODH protein;
[0191] (b) other reagents; and
[0192] (c) directions for use of said kit.
[0193] Examples of the labeled immunochemically reactive component
of such a test kit can be selected from the group consisting of
polyclonal antibodies to PRODH, monoclonal antibodies to PRODH,
chimeric antibodies to PRODH, fragments of such antibodies, and
mixtures of such antibodies.
[0194] Furthermore, the present invention extends to a test kit to
facilitate diagnosis and treatment of schizophrenia or a disease or
disorder related thereto in a subject, wherein the test kit
comprises:
[0195] (a) PCR oligonucleotide primers suitable to detecting a
variant allele of the PRODH gene in a sample;
[0196] (b) other reagents; and
[0197] (c) directions for use of the kit.
[0198] The present invention further extends to a test kit to
facilitate diagnosis and treatment of schizophrenia or a disease or
disorder related thereto in a eukaryotic cellular sample, wherein
the test kit comprises:
[0199] (a) PCR oligonucleotide primers suitable for detection of an
isolated variant allele of a PRODH gene, wherein the PRODH gene
comprises a DNA sequence of SEQ ID NO:1, and the isolated variant
comprises a DNA sequence comprising a T to C transition in the
first position of codon 497 of SEQ ID NO:1;
[0200] (b) other reagents; and
[0201] (c) directions for use of the kit.
[0202] In another embodiment, the present invention extends to
treating schizophrenia or a disease or disease or disorder related
thereto in a subject, such as obsessive compulsive disorder (OCD),
bipolar disorder (BP) or major depressive disorder. An example of
such a method comprises administering to the subject a
therapeutically effective amount of a composition comprising PRODH,
wherein PRODH comprises an amino acid sequence of SEQ ID NO:2,
conservative variants thereof, fragments thereof, or analogs or
derivatives thereof. Optionally, the composition of the invention
can be administered alone or in combination with additional
therapeutic agents to treat the subject.
[0203] In addition, the present invention extends to a method for
determining the schizophrenic-related pharmacological activity of
an agent, wherein the method comprises the steps of:
[0204] administering the agent to a mammal;
[0205] determining the level of activity of PRODH in the mammal;
and
[0206] comparing the level of activity of PRODH in the mammal to
the level of activity of PRODH in a control mammal to which the
agent was not administered. An increase in the level of activity of
PRODH in the mammal relative to activity of PRODH in the control
mammal indicates the agent has a schizophrenic-related
pharmacological activity, and potential as a therapeutic agent for
treating schizophrenia or a disease or disorder related thereto,
such as obsessive compulsive disorder (OCD), bipolar disorder (BP)
or major depressive disorder.
[0207] In yet another aspect, the present invention extends to a
method for determining the schizophrenic-related pharmacological
activity of an agent, wherein the method comprises the steps
of:
[0208] determining a basal level of activity of PRODH in the
mammal;
[0209] administering the agent to the mammal;
[0210] determining the level of activity of PRODH in the mammal
after administration of the agent; and
[0211] comparing the level of activity of PRODH after
administration of the agent to the basal level of activity.
[0212] An increase in the level of activity of PRODH in the mammal
relative to the basal level in the mammal indicates the compound
has a schizophrenic-related pharmacological activity, and may have
potential as a therapeutic agent for treating schizophrenia or a
disease or disorder related thereto, such as obsessive compulsive
disorder (OCD), bipolar disorder (BP) or major depressive
disorder.
[0213] What's more, the present invention extends to an isolated
variant allele of the Prodh gene which encodes a mutated murine
Prodh protein. In particular, the present invention extends to an
isolated variant allele of the Prodh gene, wherein the isolated
variant allele comprises a DNA sequence of FIG. 10 (SEQ ID NO:7),
degenerate variants thereof, fragments thereof, or analogs or
derivatives thereof.
[0214] The present invention also extends to an isolated nucleic
acid molecule hybridzable under standard hybridization conditions
to the isolated variant allele of the murine Prodh gene comprising
a DNA sequence of SEQ ID NO:7, degenerate variants thereof,
fragments thereof, or analogs or derivatives thereof.
[0215] In addition, the present invention extends to an isolated
variant allele of the Prodh gene which comprises a DNA sequence of
SEQ ID NO:7, degenerate variants thereof, fragments thereof, or
analogs or derivatives thereof, detectably labeled. Naturally, the
present invention extends to an isolated nucleic acid molecule
detectably labeled, wherein the isolated nucleic acid molecule is
hybridizable under standard hybridization conditions to an isolated
variant allele of the Prodh gene, wherein the isolated variant
allele comprises a DNA sequence of SEQ ID NO:7, degenerate variants
thereof, fragments thereof, or analogs or derivatives thereof.
[0216] Moreover, the present invention extends to an isolated
nucleic acid molecule encoding an isolated mutant murine Prodh
protein comprising an amino acid sequence of FIG. 11 (SEQ ID NO:8),
conservative variants thereof, fragments thereof, or analogs or
derivatives thereof.
[0217] Naturally, the present invention extends to an isolated
Prodh comprising an amino acid sequence of SEQ ID NO:8,
conservative variants thereof, fragments thereof, or analogs or
derivatives thereof.
[0218] Also, the present invention extends to an antibody having an
isolated mutant Prodh of the invention, conservative variants
thereof, fragments thereof, or analogs or derivatives thereof as an
immunogen. Such an antibody can be polyclonal, monoclonal, or
chimeric. Further, such an antibody can be detectably labeled. As
explained above, numerous examples of detectable labels having
applications in an antibody of the invention are described
infra.
[0219] In addition, the present invention extends to cloning
vectors for creating copies or "cloning" an isolated variant allele
of a Prodh gene of the invention, degenerate variants thereof,
fragments thereof, or analogs or derivatives thereof. In
particular, the present invention extends to a cloning vector
comprising an isolated variant allele of a Prodh gene, wherein the
isolated variant allele comprises a DNA sequence of SEQ ID NO:7,
degenerate variants thereof, fragments thereof, or analogs or
derivatives thereof, and an origin of replication. In another
embodiment, the invention extends to a cloning vector comprising an
origin of replication and an isolated nucleic acid molecule
hybridizable under standard hybridization conditions to an isolated
variant allele of a Prodh gene, wherein the isolated variant allele
comprises a DNA sequence of SEQ ID NO:7, degenerate variants
thereof, fragments thereof, or analogs or derivatives thereof.
[0220] Numerous cloning vectors which are commercially available to
the skilled artisan, and can be used as a cloning vector for an
isolated variant allele of a Prodh gene. Examples of such cloning
vectors, and routine recombinant DNA techniques to produce such a
vector are described infra.
[0221] Naturally, the present invention extends to an expression
vector for expressing an isolated variant allele of a murine Prodh
gene, degenerate variants thereof, fragments thereof, or analogs or
derivatives thereof, along with an isolated nucleic acid molecule
hybridizable thereto under standard hybridization conditions, to
produce a mutated murine Prodh protein, conservative variants
thereof, fragments thereof, or analogs or derivatives thereof. In
particular, an expression vector of the invention comprises an
isolated variant allele of the murine Prodh gene, wherein the
isolated variant allele comprises a DNA sequence of SEQ ID NO:7,
degenerate variants thereof, fragments thereof, or analogs or
derivatives thereof, operatively associated with a promoter. In
another embodiment, an expression vector of the invention comprises
an isolated nucleic acid molecule operatively associated with a
promoter, wherein the isolated nucleic acid molecule is hybridzable
under standard hybridization conditions to an isolated variant
allele of the Prodh gene, wherein the isolated variant allele
comprises a DNA sequence of SEQ ID NO:7, degenerate variants
thereof, fragments thereof, or analogs or derivatives thereof.
[0222] Numerous expression vectors can be used to express the
isolated variant allele of the Prodh gene, degenerate variants
thereof, fragments thereof, or analogs or derivatives thereof, or
an isolated nucleic acid molecule hybridizable to the isolated
variant allele under standard hybridization conditions. In
particular, such expression vectors are generally commercially
available to the skilled artisan, and like cloning vectors,
comprise polylinker sites. As a result, commercially available
expression vectors can be manipulated in a similar fashion in which
cloning vectors of the invention are manipulated. Hence a skilled
artisan can readily insert the isolated variant allele of the Prodh
gene, degenerate variants thereof, fragments thereof, or analogs or
derivatives thereof, or an isolated nucleic acid molecule
hybridizable thereto under standard hybridization conditions into
an expression vector such that the isolated variant allele or an
isolated nucleic acid molecule hybridizable thereto under standard
hybridization conditions is operatively associated with a promoter.
Examples of expression vectors having applications herein are
described infra.
[0223] Moreover, the present invention extends to a unicellular
host transformed or transfected with an expression vector
comprising an isolated variant allele of the Prodh gene,
operatively associated with a promoter, wherein the isolated
variant allele encodes a mutant murine proline dehydrogenase
protein comprising an amino acid sequence of SEQ ID NO:8,
conservative variants thereof, fragments thereof, or analogs or
derivatives thereof. In one embodiment, the present invention
extends to a unicellular host transformed or transfected with an
expression vector comprising an isolated variant allele of the
Prodh gene, operatively associated with a promoter, wherein the
isolated variant allele comprises a DNA sequence of SEQ ID NO:7,
degenerate variants thereof, fragments thereof, or analogs or
derivatives thereof. In another embodiment, the invention extends
to a unicellular host transformed or transfected with an expression
vector comprising an isolated nucleic acid molecule operatively
associated with a promoter, wherein the isolated nucleic acid
molecule is hybridizable under standard hybridization conditions to
an isolated variant allele of the Prodh gene, wherein the isolated
variant allele comprises a DNA sequence of SEQ ID NO:7, a
degenerate variant thereof, a fragment thereof, or an analog or
derivative thereof. Numerous unicellular hosts which are readily
available to the skilled artisan have applications in the present
invention.
[0224] Naturally, the present invention extends to method for
producing a mutant murine proline dehydrogenase protein comprising
an amino acid sequence of SEQ ID NO:8, conservative variants
thereof, fragments thereof, or analogs or derivatives thereof. In
one embodiment, a method for producing a mutant murine proline
dehydrogenase comprises the steps of:
[0225] a) culturing a unicellular host of transformed or
transfected with an expression vector comprising an isolated
variant allele of the prodh gene, wherein the isolated variant
allele comprises a DNA sequence of SEQ ID NO:7, a degenerate
variant thereof, a fragment thereof, or an analog or derivative
thereof, operatively associated with a promoter, under conditions
that provide for expression of the isolated variant allele to
produce a mutant murine proline dehydrogenase protein comprising an
amino acid sequence of SEQ ID NO:8, a conservative variant thereof,
a fragment thereof, or analog or derivative thereof; and
[0226] b) recovering the protein from the unicellular host, the
culture, or both.
[0227] In another embodiment, the method comprises the steps
of:
[0228] a) culturing a unicellular host of transformed or
transfected with an expression vector comprising an isolated
nucleic acid molecule hybridizable under standard hybridization
conditions to an isolated variant allele of the Prodh gene, wherein
the isolated variant allele comprises a DNA sequence of SEQ ID
NO:7, a degenerate variant thereof, a fragment thereof, or an
analog or derivative thereof, operatively associated with a
promoter, under conditions that provide for expression of the
isolated nucleic acid molecule, to produce a protein comprising an
amino acid sequence of SEQ ID NO:8, a conservative variant thereof,
a fragment thereof, or an analog or derivative thereof, and
[0229] b) recovering the protein from the unicellular host, the
culture, or both.
[0230] Furthermore, the present invention extends to a method for
identifying a drug or agent for treating schizophrenia or a disease
or disorder related thereto. An example of such a method comprises
the steps of:
[0231] performing an first pre-pulse inhibition test (PPI) test on
a mouse having within its genome two copies of an isolated variant
allele of a Prodh gene comprising a DNA sequence of SEQ ID NO:7,
wherein both copies are capable of expressing a mutant Prodh
comprising an amino acid sequence of SEQ ID NO:8, to obtain a first
percentage of inhibition of startle response;
[0232] administering the potential drug or agent to the mouse;
[0233] performing a second PPI test on the mouse to obtain a second
percentage of inhibition of startle response; and
[0234] comparing the first percentage to the inhibition of startle
response with the second percentage of startle response,
[0235] wherein an increase in percentage of inhibition in the
second percentage of inhibition relative to the first percentage of
inhibition is indicative of the ability of the drug or agent to
treat schizophrenia or a disease or disorder related thereto. Thus,
if the percentage of inhibition of startle response in the mouse
having within its two active copies of an isolated variant allele
of a Prodh gene comp mouse after administration of the drug or
agent is greater than the percentage of inhibition of startle
response in the Pro/Re mouse prior to inhibition, then the drug or
agent has the ability to treat schizophrenia or a disease or
disorder related thereto, such as obsessive compulsive disorder
(OCD), bipolar disorder (BP) or major depressive disorder.
[0236] Also, the present invention extends to a method for
identifying a drug or agent for treating schizophrenia or a disease
or disorder related thereto. An example of such a method comprises
the steps of:
[0237] performing an first pre-pulse inhibition test (PPI) test on
an F3 generation mouse from a cross Pro/Re X C57B1/6J wild-type,
wherein the F3 generation mouse has two copies within its genome of
an isolated variant allele of a Prodh gene comprising a DNA
sequence of SEQ ID NO:7 which are capable of expressing a mutant
Prodh comprising an amino acid sequence of SEQ ID NO:8, to obtain a
first percentage of inhibition of startle response;
[0238] administering the potential drug or agent to the F3
generation mouse from a cross of Pro/Re X C57B1/6J wild-type;
[0239] performing a second PPI test on the F3 generation mouse from
a cross of Pro/Re X C57B1/6J wild-type to obtain a second
percentage of inhibition of startle response; and
[0240] comparing the first percentage to the inhibition of startle
response with the second percentage of startle response,
[0241] wherein an increase in percentage of inhibition in the
second percentage of inhibition relative to the first percentage of
inhibition is indicative of the ability of the drug or agent to
treat schizophrenia or a disease or disorder related thereto.
[0242] What's more, the present invention extends to a method for
identifying a drug or agent for use in treating schizophrenia or a
disease or disorder related thereto, comprising the steps of:
[0243] a) administering the drug or agent to an F3 generation mouse
from a cross of Pro/Re X C57B1/6J wild-type, wherein the F3
generation mouse has two copies within its genome of an isolated
variant allele of a Prodh gene comprising a DNA sequence of SEQ ID
NO:7 which are capable of expressing a mutant Prodh comprising an
amino acid sequence of SEQ ID NO:8;
[0244] b) performing a PPI test on the F3 generation mouse from a
cross of Pro/Re X C57B1/6J wild-type to obtain a percentage of
inhibition of the startle response in the F3 generation mouse from
a cross of Pro/Re X C57B1/6J wild-type which was administered the
drug or agent; and
[0245] c) comparing the percentage of inhibition of the startle
response in the F3 generation mouse from a cross of Pro/Re X
C57B1/6J wild-type with the percentage of inhibition of the startle
response in an unmedicated F3 generation mouse from a cross of
Pro/Re X C57B1/6J wild-type, wherein the F3 generation mouse has
two copies within its genome of an isolated variant allele of a
Prodh gene comprising a DNA sequence of SEQ ID NO:7 which are
capable of expressing a mutant Prodh comprising an amino acid
sequence of SEQ ID NO:8.
[0246] An increase in percentage of inhibition in the percentage of
inhibition in the medicated mouse relative to the percentage of
inhibition in the unmedicated mouse is indicative of the ability of
the drug or agent to treat schizophrenia or a disease or disorder
related thereto.
[0247] Furthermore, the present invention extends to a method for
identifying a drug or agent for use in treating schizophrenia or a
disease or disorder related thereto, comprising the steps of:
[0248] a) administering the drug or agent to a mouse having within
its genome two copies of an isolated variant allele of a Prodh gene
comprising a DNA sequence of SEQ ID NO:7, wherein both copies are
capable of expressing a mutant Prodh comprising an amino acid
sequence of SEQ ID NO:8;
[0249] b) performing a PPI test on the mouse to obtain a percentage
of inhibition of the startle response in the mouse; and
[0250] c) comparing the percentage of inhibition of the startle
response in the mouse with the percentage of inhibition of the
startle response in an unmedicated mouse having within its genome
two copies of an isolated variant allele of a Prodh gene comprising
a DNA sequence of SEQ ID NO:7, wherein both copies are capable of
expressing a mutant Prodh comprising an amino acid sequence of SEQ
ID NO:8.
[0251] An increase in percentage of inhibition in the percentage of
inhibition in the medicated mouse relative to the percentage of
inhibition in the unmedicated mouse is indicative of the ability of
the drug or agent to treat schizophrenia or a disease or disorder
related thereto.
[0252] In addition, the present invention extends to a method for
identifying a drug or agent for use in treating schizophrenia or a
disease or disorder related thereto, comprising the steps of:
[0253] a) administering the drug or agent to a mouse having within
its genome two copies of an isolated variant allele of a Prodh gene
comprising a DNA sequence of SEQ ID NO:7, wherein both copies are
capable of expressing a mutant Prodh comprising an amino acid
sequence of SEQ ID NO:8;
[0254] b) performing a PPI test on the mouse to obtain a percentage
of inhibition of the startle response in the mouse; and
[0255] c) comparing the percentage of inhibition of the startle
response in the mouse with the percentage of inhibition of the
startle response in an unmedicated mouse having within its genome
two copies of an isolated Prodh gene comprising a DNA sequence of
SEQ ID NO:3, wherein both copies are capable of expressing a Prodh
comprising an amino acid sequence of SEQ ID NO:4.
[0256] If the percentage of inhibition of the startle response in
the medicated mouse is statistically equivalent to the percentage
of inhibition in the mouse capable of expressing Prodh comprising a
DNA sequence of SEQ ID NO:4, then the drug or agent has the ability
to treat schizophrenia or a disease or disorder related
thereto.
[0257] In another embodiment, the present invention extends to an a
method for identifying a drug or agent for use in treating
schizophrenia or a disease or disorder related thereto, comprising
the steps of:
[0258] a) administering the drug or agent to an F3 generation mouse
from a cross of Pro/Re X C57B1/6J wild-type, wherein the F3
generation mouse has two copies within its genome of an isolated
variant allele of a Prodh gene comprising a DNA sequence of SEQ ID
NO:7 which are capable of expressing a mutant Prodh comprising an
amino acid sequence of SEQ ID NO:8;
[0259] b) performing a PPI test on the F3 generation mouse from a
cross of Pro/Re X C57B1/6J wild-type administered the drug or agent
to obtain a percentage of inhibition of the startle response in the
mouse; and
[0260] c) comparing the percentage of inhibition of the startle
response in F3 generation mouse from a cross of Pro/Re X C57B1/6J
wild-type administered the drug with the percentage of inhibition
of the startle response in an F3 generation mouse from a cross of
Pro/Re X C57B1/6J wild-type, wherein the F3 generation mouse has
two copies within its genome of an isolated Prodh gene comprising a
DNA sequence of SEQ ID NO:3 which are capable of expressing a Prodh
comprising an amino acid sequence of SEQ ID NO:4.
[0261] If the percentage of inhibition of the startle response in
the medicated mouse is statistically equivalent to the percentage
of inhibition in the mouse capable of expressing Prodh comprising a
DNA sequence of SEQ ID NO:4, then the drug or agent has the ability
to treat schizophrenia or a disease or disorder related
thereto.
[0262] The PPI Test is Described infra.
[0263] Accordingly it is an object of the invention to provide the
DNA sequences of murine and human proline dehydrogenase genes, and
the amino acid sequences of murine and human proline
dehydrogenase.
[0264] It is another object of the present invention to provide
heretofore unknown variant alleles of the human PRODH gene, which
can be used to map the locus of the human PRODH gene.
[0265] It is another object of the invention to provide a
heretofore unknown variant allele of the human PODH gene which is a
marker for a susceptibility to, or the presence of schizophrenia or
a disease or disorder related thereto in a subject, such as
obsessive compulsive disorder (OCD), bipolar disorder (BP) or major
depressive disorder in a subject.
[0266] It is yet another object of the present invention to provide
isolated nucleic acid molecules, optionally detectably labeled,
which are hybridizable under standard hybridization conditions to
variant alleles of the PRODH gene disclosed herein.
[0267] It is yet another object of the present invention to provide
variant PRODH proteins, produced from the expression of a variant
alleles of the human PRODH gene, or isolated nucleic acid molecules
hybridizable to such variant alleles under standard hybridization
conditions.
[0268] It is yet another object of the present invention to provide
antibodies, optionally detectably labeled, having a variant PRODH
protein of the present invention as an immunogen, wherein such
antibodies may be polyclonal, monoclonal or chimeric.
[0269] It is yet another object of the present invention to provide
commercial test kits for attending medical professionals to
determine the presence of a variant allele of the PRODH gene in a
bodily sample taken from a subject. The results of such testing can
then be used to determine the subject's susceptibility to suffer
from schizophrenia or a disease or disorder related thereto, such
as obsessive compulsive disorder (OCD), bipolar disorder (BP) or
major depressive disorder, or to diagnose such a disease or
disorder.
[0270] It is yet another object of the present invention to provide
a method and associated assay system for screening subjects in
order to determine their susceptibility to schizophrenia or a
disease or disorder related thereto, and to likewise select an
appropriate course of therapy therefor.
[0271] It is yet another object of the present invention to provide
compositions such as drugs, agents and the like, potentially
effective in either potentiating the effects of PRODH, or
increasing levels of PRODH in mammalian, especially human
patients.
[0272] It is still yet another object of the present invention to
provide a method for the treatment of mammals to modulate the
amount or activity of PRODH or subunits thereof in the mammal, so
as to alter the adverse consequences of diminished levels of PRODH,
which can result in schizophrenia or a disease or disorder related
thereto, such as obsessive compulsive disorder (OCD), bipolar
disorder (BP) or major depressive disorder.
[0273] It is a still yet another object of the present invention to
provide pharmaceutical compositions for use in therapeutic methods
which comprise or are based upon the PRODH, its subunits, their
binding partner(s), as well as molecules whose activity or
production depends on PRODH; or upon molecules or agents or drugs
that control the production, stability and degradation, or that
mimic the activities of the PRODH.
[0274] It is yet still another object of the present invention to
provide methods of determining a susceptibility for, or presence
of, schizophrenia, or a disease or disorder related thereto, by
determining the levels of an enzyme involved in proline catabolism,
wherein such an enzyme comprises PRODH.
[0275] It is yet still another object of the present invention to
provide numerous methods for the selection of a drug or therapeutic
agent to treat potentially schizophrenia, or a disease or disorder
related thereto, such as obsessive compulsive disorder (OCD),
bipolar disorder (BP) or major depressive disorder.
[0276] It is yet still another object of the invention to provide
the DNA sequence of the wild-type murine Prodh gene, and an
isolated variant of the allele of the wild-type murine Prodh
gene.
[0277] It is yet still another object of the invention to provide
the amino acid sequence of wild-type murine Prodh protein, as well
as a mutant murine Prodh protein.
[0278] It is yet still another object of the invention to provide
mammalian assays for determining whether a drug or agent has the
ability to treat schizophrenia or a disease or disorder related
thereto. Such assays involve Pro/Re mice described in the Example,
and the PPI test, described below.
[0279] These and other aspects of the present invention will be
better appreciated by reference to the following drawings and
Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0280] FIG. 1: (A) Multiple alignment of the predicted protein
sequences of H. sapiens, M. musculus, D. melanogaster and S.
cerevisiae proline dehydrogenase. Shaded areas highlight
identities. Also, an asterisk (Q) indicates a mutation in murine
Prodh. (B) Localization of the human proline dehydrogenase gene to
chromosome 22q11: Southern blot hybridization analysis of an array
of nine PACs mapped and ordered within the 22q11 microdeletion
[Carlson et al., 1997]. A human PRODH cDNA fragment was used as a
hybridization probe. Only PAC-P457M14 (Q) provided a positive
signal. The positions of the COMT and DGCR6 genes, previously
mapped in this region [M. H. Grossman, B. S. Emanuel, M. L. Budarf,
Genomics 12. 822 (1992); S. Demczuk, G. Thomas, A. Aurias, Hum.
Mol. Genet. 5, 633 (1996)] are indicated as reference points.
Markers D22S427 and D22S264, that flank the smallest 22q11 deletion
associated with schizophrenia as presented in [Karayiorgou et al.,
1995], are also indicated. (C) High resolution Northern blot
analysis of the brain expression pattern of human PRODH (human
brain mRNA filter was purchased from Clontech (Palo Alto, Calif.));
hybridization to a human .beta.-actin probe was used to confirm
equal loading of undegraded mRNA in each lane.
[0281] FIG. 2: (A) Northern blot analysis of expression of the
mouse Prodh gene. The size of the markers in kilobases are
indicated to the left of the blot. (B) Mutational analysis of the
mouse Prodh gene from the Pro/Re strain: segment of antisense
genomic sequence including the G-->T (C-->A) substitution
(arrow) identified 135 base pairs upstream of the native
termination codon, introducing a premature translational
termination. (C) Analysis of plasma proline and glutamate levels in
homozygous and wild type F3 generation mice from a cross between
the original Pro/Re and C57/B6 wild type strain. The presence of
the identified mutation correlates with increased levels of proline
in F3 mice (p<0.0001). Y-axis values represent mmoles/L
(.+-.SEM). (D) Basal levels of glutamate, GABA and aspartate in the
brains of Prodh-deficient mice and littermate controls.
Neurotransmitter levels were analyzed one week after the
termination of the behavioral analysis. Panels show basal levels of
glutamate, GABA and aspartate in the frontal cortex, hypothalamus,
amygdala, and hippocampus of homozygous and wild type animals of
both sexes. Y-axis values are mean (.+-.S.E.M). GAG/GAG wild type
mice (gray bar); TAG/TAG homozygous mice (solid bar). Data was
analyzed by two-way ANOVA (*p<0.05, ****p<0.0001).
[0282] FIG. 3: Sensorimotor gating in Prodh-deficient mice: (A)
Schematic outline of the experiment. (B) Prepulse inhibition of an
acoustic startle response; prepulse inhibition was examined for a
combination of two startle dB levels (110 dB and 115 dB) and two
prepulse dB levels (82 dB and 90 dB); higher Y-axis values (mean
.+-.SEM) represent greater percent inhibition. ANOVA with repeated
measures revealed a significant attenuation in the overall level of
PPI in the homozygous mutant mice compared to wild type littermates
[F(1,66)=6.14, p=0.015]. (C) Amplitude of startle response at two
different startling stimuli; Y-axis values (mean .+-.SEM) represent
weight-corrected peak amplitude startle (Vmax). No significant
differences were observed between genotypes at either 100 dB
(p=0.0799) or 115 dB (p=0.125). (D) Habituation of the startle
response to repeated presentations of an 115 dB burst.
Weight-corrected startle magnitude values (Vmax) were averaged,
expressed as a percentage of the first block and analyzed using
ANOVA with repeated measures. No significant differences were
observed between genotypes (p=0.675).
[0283] FIG. 4: Locomotion and anxiety-like behaviors (collectively
termed anxiety, reactivity or emotionality) of Prodh-deficient
mice: (A) Open field locomotion assay: No significant differences
between genotypes were observed in total distance traveled
(p=0.747), stereotypic behavior (p=0.839), or time spent at the
margin of the field (an estimate of anxiety, p=0.078). Data were
collected every 5 minutes over a 15 minute period and analyzed with
ANOVA with repeated measures. Because animals were not preexposed
to the chamber prior to testing, data collected every five minutes
were also analyzed separately using two-way ANOVA (not shown) but
no genotype or sex effect were observed. (B) Dark-light assay: no
significant differences were observed between genotypes in latency
to emerge into the lit compartment (p=0.597), or in horizontal
activity in either the lit (p=0.194) or the dark compartment
(p=0.711).
[0284] FIG. 5: shows the results of the testing of families with
schizophrenia to determine whether the polymorphism of the PRODH
gene comprising a variation in SEQ ID NO:1, wherein the variation
comprises a silent T to C transition at the first position of codon
497, which introduces a PuvII site is preferentially transmitted in
such families. In particular, results of a Transmission
Disequilibrium Test (TDT) are disclosed which show the preferential
transmission of a variant allele of the PRODH gene comprising a DNA
sequence having a variation in SEQ ID NO:1, wherein the variation
comprises a silent T to C transition at the first position of codon
497.
[0285] FIG. 6 shows the human PRODH cDNA sequence (SEQ ID NO:1).
Underlined nucleotides 1-3 make up the first codon of the sequence,
and underlined nucleotides 1549-1551 make up the termination
codon.
[0286] FIG. 7 shows the amino acid sequence of human proline
dehydrogenase (SEQ ID NO:2).
[0287] FIG. 8 shows the murine Prodh cDNA sequence (SEQ ID
NO:3).
[0288] FIG. 9 shows the amino acid sequence of murine proline
dehydrogenase (SEQ ID NO:4).
[0289] FIG. 10 shows the DNA sequence of a variant allele of the
murine Prodh gene (SEQ ID NO:7).
[0290] FIG. 11 shows the amino acid sequence of mutant murine Prodh
protein (SEQ ID NO:8).
[0291] FIG. 12 shows the DNA sequence of an isolated nucleic acid
molecule of the invention which encodes human proline dehydrogenase
(SEQ ID NO:9). Underlined nucleotides 447-449 make up the first
codon, and underlined nucleotides 1995-1997 make up the termination
codon.
DETAILED DESCRIPTION OF THE INVENTION
[0292] As explained above, the present invention is based upon the
discovery of the DNA sequences of human PRODH and murine Prodh, a
heretofore unknown variant of murine Prodh which encodes a mutant
Prodh protein, and heretofore unknown variant alleles of the PRODH
gene, that encode heretofore unknown variant human proline
dehydrogenase (PRODH) proteins.
[0293] In addition, the present invention is based upon Applicants'
discovery that unexpectedly, a correlation exists between the
presence of schizophrenic symptoms in a subject, and the presence
of a variant allele of the PRODH gene in the subject's genome.
Hence, a variant allele of human PRODH can serve as a genetic
marker to determine a susceptibility to, or presence of
schizophrenia or a disease or disorder related thereto, such as
obsessive compulsive disorder (OCD), bipolar disorder (BP) or major
depressive disorder (MDD) in a subject.
[0294] Furthermore, the present invention extends to diagnostic
methods to determine a subject's increased or decreased
susceptibility to schizophrenia or a disease or disorder related
thereto. With the results of such methods, targeted prevention
methods, early therapeutic intervention, and improved chronic
treatment for schizophrenia or a disease or disorder related
thereto are set forth herein and encompassed by the present
invention. In addition, attending medical professionals armed with
the results of such diagnostic methods can determine an appropriate
regimen to treat the subject.
[0295] What's more, the present invention extends to assays to
determine the ability of a drug or agent can be used to treat
schizophrenia or a disease or disorder related thereto.
[0296] Numerous terms and phrases are used throughout the instant
Application and are defined below:
[0297] As used herein "PRODH" refers to a wild type human proline
dehydrogenase gene, and "PRODH" refers to a wild-type human proline
dehydrogenase protein. An example of the PRODH gene comprises a DNA
sequence of SEQ ID NO:1. Another example comprises the DNA sequence
of SEQ ID NO:9.
[0298] As used herein, the phrase "isolated variant allele of a
human proline dehydrogenase (PRODH) gene" refers to an assembly of
nucleotides that includes cDNA and genomic DNA nucleic acids, which
is a mutational state of the wild-type PRODH gene.
[0299] As used herein, an "isolated human variant proline
dehydrogenase" or a "variant PRODH" refer to a protein which is a
mutational state of the wild type proline PRODH.
[0300] As used herein, "Prodh" refers to a wild-type murine proline
dehydrogenase gene, and "Prodh" refers to a wild-type murine
proline dehydrogenase protein.
[0301] As used herein, the phrase "isolated variant allele of
murine Prodh gene" refers to an assembly of nucleotides that
includes cDNA and genomic DNA nucleic acids, which is a mutational
state of the wild-type Prodh gene. A particular example of an
isolated variant allele of the murine Prodh gene comprises a DNA
sequence of FIG. 10 (SEQ ID NO:7).
[0302] As used herein, the phrase "mutant Prodh protein" or
"variant Prodh" refers to a protein encoded by an isolated variant
allele of murine Prodh gene, which has an amino acid sequence that
is different from the amino acid sequence of wild-type murine Prodh
protein. A particular example of wild-type murine Prodh protein
comprises an amino acid sequence of FIG. 9 (SEQ ID NO:4), while a
mutant murine Prodh protein comprises an amino acid sequence of
FIG. 11 (SEQ ID NO:8). A comparison of these two sequences shows
that the mutant murine Prodh has an amino acid sequence different
from that of Prodh.
[0303] As used herein, the term "transition" refers to a mutational
event in which one purine is replaced by another, or one pyrimidine
is replaced by another.
[0304] As used herein, the term "codon" refers to a triplet of
bases in a DNA or RNA molecule that specifies or encodes the
information for a single amino acid.
[0305] As used herein, the phrase "F3 generation mouse from a cross
of Pro/Re X C57B1/6J wild-type, wherein the F3 generation mouse has
two copies within its genome of an isolated variant allele of a
Prodh gene comprising a DNA sequence of SEQ ID NO:7 which are
capable of expressing a mutant Prodh comprising an amino acid
sequence of SEQ ID NO:8" refers to the third generation mouse of a
line of mice formed from a cross between the Pro/Re strain of mice
and the wild-type C57B1/6J wild-type strain, wherein the mouse is
homozygous for the variant allele of Prodh comprising the DNA
sequence of SEQ ID NO:7, and expresses the variant Prodh comprising
a DNA sequence of SEQ ID NO:8.
[0306] As used herein, the phrase "F3 generation mouse from a cross
of Pro/Re X C57B1/6J wild-type, wherein the F3 generation mouse has
two copies within its genome of an isolated Prodh gene comprising a
DNA sequence of SEQ ID NO:3 which are capable of expressing a Prodh
comprising an amino acid sequence of SEQ ID NO:4" refers to the
third generation mouse of a line of mice formed from a cross
between the Pro/Re strain of mice and the wild-type C57BL/6J
wild-type strain, wherein the mouse is homozygous for the Prodh
gene comprising the DNA sequence of SEQ ID NO:3, and expresses
Prodh comprising a DNA sequence of SEQ ID NO:4.
[0307] As used herein, the terms "schizophrenia", "obsessive
compulsive disorder", "bipolar disorder", and "major depressive
disorder" refer to psychiatric diseases or disorders that are
readily understood by the skilled artisan and are set forth in
American Psychiatric Associate (1994); Diagnostic and Statistical
Manual of Mental Disorders, 4.sup.th Edition. Washington, D.C.
[0308] As used herein, the term "susceptibility" to schizophrenia
or a disease or disorder related thereto refers to a subject's
potential of being affected with such a disease or disorder.
[0309] As used herein, the terms "standard" and "control" refer to
a bodily sample, cell extract, etc. established for use as a rule
or basis of comparison in measuring levels of activity of PRODH,
results of PPI, etc.
[0310] As used herein, the term "combination" referring to
variations in the wild-type sequence, either amino acid residues or
nucleotides, indicates that two or more of the discovered
variations in the particular DNA sequence of amino acid sequence
can be present in variant allele of the wild-type nucleic acid
molecule or protein.
[0311] An initial aspect of the invention extends to an isolated
nucleic acid molecule comprising a DNA sequence of SEQ ID NO:1,
degenerate variants thereof, fragments thereof, or analogs or
derivatives thereof.
[0312] Another aspect of the invention extends to an isolated
nucleic acid molecule comprising a DNA sequence of SEQ ID NO:3,
degenerate variants thereof, fragments thereof, or analogs or
derivatives thereof.
[0313] Another aspect of the present invention extends an isolated
variant allele of the PRODH gene, wherein the PRODH gene comprises
a DNA sequence of SEQ ID NO:1 or SEQ ID NO:9, and an isolated
variant allele of the PRODH comprises a DNA sequence having at
least one variation in SEQ ID NO:1 or SEQ ID NO:9, wherein the at
least one variation comprises:
[0314] a G to A transition in the third position of codon 83;
[0315] a C to T transition in the first position of codon 101;
[0316] a G to A transition in the second position of codon 101;
[0317] a C to T transition in the first position of codon 247;
[0318] a C to T transition in the third position of codon 342;
[0319] a C to T transition in the third position of codon 421;
[0320] an A to G transition in the second position of codon
437;
[0321] a T to C transition in the first position of codon 497;
or
[0322] a combination thereof.
[0323] Furthermore, the present invention is based on discovery
that surprisingly and unexpectedly, a particular variant allele of
the PRODH gene is present in a statistically significantly higher
frequency in subjects suffering from a psychiatric disease or
disorder.
[0324] Further explanation of this aspect of the invention is set
forth infra.
[0325] Consequently, an initial aspect of the present invention
contemplates isolation of PRODH, Prodh, and heretofore unknown
variant alleles of the human PRODH gene. As used herein, the term
"gene" refers to an assembly of nucleotides that encode a
polypeptide, and includes cDNA and genomic DNA nucleic acids.
[0326] Furthermore, in accordance with the present invention there
may be employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory
Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (herein "Sambrook et al., 1989"); DNA
Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed.
1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic
Acid Hybridization [B. D. Hames & S. J. Higgins eds. (1985)];
Transcription And Translation [B. D. Hames & S. J. Higgins.
[0327] eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed.
(1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B.
Perbal, A Practical Guide To Molecular Cloning (1984); F. M.
Ausubel et al. (eds.), Current Protocols in Molecular Biology, John
Wiley & Sons, Inc. (1994).
[0328] Therefore, if appearing herein, the following terms shall
have the definitions set out below.
[0329] A "vector" is a replicon, such as plasmid, phage or cosmid,
to which another DNA segment may be attached so as to bring about
the replication of the attached segment. A "replicon" is any
genetic element (e.g., plasmid, chromosome, virus) that functions
as an autonomous unit of DNA replication in vivo, i.e., capable of
replication under its own control.
[0330] A "cassette" refers to a segment of DNA that can be inserted
into a vector at specific restriction sites. The segment of DNA
encodes a polypeptide of interest, and the cassette and restriction
sites are designed to ensure insertion of the cassette in the
proper reading frame for transcription and translation.
[0331] "Heterologous" DNA refers to DNA not naturally located in
the cell, or in a chromosomal site of the cell. Preferably, the
heterologous DNA includes a gene foreign to the cell.
[0332] As used herein, the term "wild-type" refers to the most
commonly observed phenotype or genotype, designated as the
norm.
[0333] A "nucleic acid molecule" refers to the phosphate ester
polymeric form of ribonucleosides (adenosine, guanosine, uridine or
cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine,
deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"),
or any phosphoester analogs thereof, such as phosphorothioates and
thioesters, in either single stranded form, or a double-stranded
form comprising coding and complementary strands. Double stranded
DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic
acid molecule, and in particular DNA or RNA molecule, refers only
to the primary and secondary structure of the molecule, and does
not limit it to any particular tertiary forms. Thus, this term
includes double-stranded DNA found, inter alia, in linear or
circular DNA molecules (e.g., restriction fragments), plasmids, and
chromosomes. In discussing the structure of particular
double-stranded DNA molecules, sequences may be described herein
according to the normal convention of giving only the sequence in
the 5' to 3' direction along the nontranscribed strand of DNA
(i.e., the strand having a sequence homologous to the mRNA). A
"recombinant DNA molecule" is a DNA molecule that has undergone a
molecular biological manipulation.
[0334] A nucleic acid molecule is "hybridizable" to another nucleic
acid molecule, such as a cDNA, genomic DNA, or RNA, when a single
stranded form of the nucleic acid molecule, e.g. either the coding
strand or the strand complementary to the coding strand, can anneal
to the other nucleic acid molecule under the appropriate conditions
of temperature and solution ionic strength (see Sambrook et al.,
supra). The conditions of temperature and ionic strength determine
the "stringency" of the hybridization. For preliminary screening
for homologous nucleic acids, low stringency hybridization
conditions, corresponding to a T.sub.m of 50.degree., can be used,
e.g., 5.times. SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30%
formamide, 5.times. SSC, 0.5% SDS). Moderate stringency
hybridization conditions correspond to a higher T.sub.m, e.g., a
T.sub.m of 55.degree. C., 40% formamide, with 5.times. or 6.times.
SSC. High stringency hybridization conditions correspond to the
highest T.sub.m, e.g., a T.sub.m of 60-65.degree. C., 50%
formamide, 5.times. or 6.times. SSC. Hybridization requires that
the two nucleic acids contain complementary sequences, although
depending on the stringency of the hybridization, mismatches
between bases are possible. The appropriate stringency for
hybridizing nucleic acids depends on the length of the nucleic
acids and the degree of complementation, variables well known in
the art. The greater the degree of similarity or homology between
two nucleotide sequences, the greater the value of T.sub.m for
hybrids of nucleic acids having those sequences. The relative
stability (corresponding to higher T.sub.m) of nucleic acid
hybridizations decreases in the following order: RNA:RNA, DNA:RNA,
DNA:DNA. For hybrids of greater than 100 nucleotides in length,
equations for calculating T.sub.m have been derived (see Sambrook
et al., supra, 9.50-9.51). For hybridization with shorter nucleic
acids, i.e., oligonucleotides, the position of mismatches becomes
more important, and the length of the oligonucleotide determines
its specificity (see Sambrook et al., supra, 11.7-11.8). Preferably
a minimum length for a hybridizable nucleic acid is at least about
10 nucleotides; particularly at least about 15 nucleotides; more
particularly at least about 20 nucleotides; even more particularly
at least about 30 nucleotides, and yet more particularly at least
about 40 nucleotides, and most particularly about 50
nucleotides.
[0335] In a specific embodiment, the term "standard hybridization
conditions" refers to a T.sub.m of 55.degree. C., and utilizes
conditions as set forth above. In a preferred embodiment, the
T.sub.m is 60.degree. C.; in a more preferred embodiment, the
T.sub.m is 65.degree. C.
[0336] "Homologous recombination" refers to the insertion of a
foreign DNA sequence of a vector in a chromosome. Preferably, the
vector targets a specific chromosomal site for homologous
recombination. For specific homologous recombination, the vector
will contain sufficiently long regions of homology to sequences of
the chromosome to allow complementary binding and incorporation of
the vector into the chromosome. Longer regions of homology, and
greater degrees of sequence similarity, may increase the efficiency
of homologous recombination.
[0337] A DNA "coding sequence" is a double-stranded DNA sequence
which is transcribed and translated into a polypeptide in a cell in
vitro or in vivo when placed under the control of appropriate
regulatory sequences. The boundaries of the coding sequence are
determined by a start codon at the 5' (amino) terminus and a
translation stop codon at the 3' (carboxyl) terminus. A coding
sequence can include, but is not limited to, prokaryotic sequences,
cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic
(e.g., mammalian) DNA, and even synthetic DNA sequences. If the
coding sequence is intended for expression in a eukaryotic cell, a
polyadenylation signal and transcription termination sequence will
usually be located 3' to the coding sequence.
[0338] Transcriptional and translational control sequences are DNA
regulatory sequences, such as promoters, enhancers, terminators,
and the like, that provide for the expression of a coding sequence
in a host cell. In eukaryotic cells, polyadenylation signals are
control sequences.
[0339] A "promoter sequence" or "promoter" is a DNA regulatory
region capable of binding RNA polymerase in a cell and initiating
transcription of a downstream (3' direction) coding sequence. For
purposes of defining the present invention, the promoter sequence
is bounded at its 3' terminus by the transcription initiation site
and extends upstream (5' direction) to include the minimum number
of bases or elements necessary to initiate transcription at levels
detectable above background. Within the promoter sequence will be
found a transcription initiation site (conveniently defined for
example, by mapping with nuclease S1), as well as protein binding
domains (consensus sequences) responsible for the binding of RNA
polymerase.
[0340] A coding sequence is "under the control" of transcriptional
and translational control sequences in a cell when RNA polymerase
transcribes the coding sequence into mRNA, which is then trans-RNA
spliced and translated into the protein encoded by the coding
sequence.
[0341] A coding sequence is "operatively associated with" a
transcriptional and translational control sequences, such as a
promoter for example, when RNA polymerase transcribes the coding
sequence into mRNA, which in turn is translated into a protein
encoded by the coding sequence.
[0342] A "signal sequence" is included at the beginning of the
coding sequence of a protein to be expressed on the surface of a
cell. This sequence encodes a signal peptide, N-terminal to the
mature polypeptide, that directs the host cell to translocate the
polypeptide. The term "translocation signal sequence" is used
herein to refer to this sort of signal sequence. Translocation
signal sequences can be found associated with a variety of proteins
native to eukaryotes and prokaryotes, and are often functional in
both types of organisms.
[0343] An "expression control sequence" is a DNA sequence that
controls and regulates the transcription and translation of another
DNA sequence. A coding sequence is "under the control" of
transcriptional and translational control sequences in a cell when
RNA polymerase transcribes the coding sequence into mRNA, which is
then translated into the protein encoded by the coding
sequence.
[0344] The term "oligonucleotide" as used herein in referring to
the probe of the present invention, is defined as a molecule
comprised of two or more ribonucleotides, preferably more than
three. Its exact size will depend upon many factors which, in turn,
depend upon the ultimate function and use of the
oligonucleotide.
[0345] The term "primer" as used herein refers to an
oligonucleotide, whether occurring naturally as in a purified
restriction digest or produced synthetically, which is capable of
acting as a point of initiation of synthesis when placed under
conditions in which synthesis of a primer extension product, which
is complementary to a nucleic acid strand, is induced, i.e., in the
presence of nucleotides and an inducing agent such as a DNA
polymerase and at a suitable temperature and pH. The primer may be
either single-stranded or double-stranded and must be sufficiently
long to prime the synthesis of the desired extension product in the
presence of the inducing agent. The exact length of the primer will
depend upon many factors, including temperature, source of primer
and use of the method. For example, for diagnostic applications,
depending on the complexity of the target sequence, the
oligonucleotide primer typically contains 15-50 or more
nucleotides, although it may contain fewer nucleotides.
[0346] The primers herein are selected to be "substantially"
complementary to different strands of a particular target DNA
sequence. This means that the primers must be sufficiently
complementary to hybridize with their respective strands.
Therefore, the primer sequence need not reflect the exact sequence
of the template. For example, a non-complementary nucleotide
fragment may be attached to the 5' end of the primer, with the
remainder of the primer sequence being complementary to the strand.
Alternatively, non-complementary bases or longer sequences can be
interspersed into the primer, provided that the primer sequence has
sufficient complementary with the sequence of the strand to
hybridize therewith and thereby form the template for the synthesis
of the extension product.
[0347] A cell has been "transfected" by exogenous or heterologous
DNA when such DNA has been introduced inside the cell. A cell has
been "transformed" by exogenous or heterologous DNA when the
transfected DNA effects a phenotypic change. Preferably, the
transforming DNA should be integrated (covalently linked) into
chromosomal DNA making up the genome of the cell.
[0348] A "clone" is a population of cells derived from a single
cell or common ancestor by mitosis. A "cell line" is a clone of a
primary cell that is capable of stable growth in vitro for many
generations.
[0349] As used herein, the phrase "an isolated nucleic acid
molecule of the invention" refers to any of the following:
[0350] a) an isolated nucleic acid molecule comprising a DNA
sequence of SEQ ID NO:1, degenerate variants thereof, fragments
thereof, or analogs or derivatives thereof;
[0351] b) an isolated nucleic acid hybridizable under standard
hybridization conditions to an isolated nucleic acid molecule
comprising a DNA sequence of SEQ ID NO:1, degenerate variants
thereof, fragments thereof, or analogs or derivatives thereof;
[0352] c) an isolated nucleic acid molecule comprising a DNA
sequence of SEQ ID NO:3, degenerate variants thereof, fragments
thereof, or analogs or derivatives thereof;
[0353] d) an isolated nucleic acid molecule hybridizable under
standard hybridization conditions to an isolated nucleic acid
molecule comprising a DNA sequence of SEQ ID NO:3, degenerate
variants thereof, fragments thereof, or analogs or derivatives
thereof,
[0354] e) an isolated variant allele of a PRODH gene, wherein the
isolated variant allele comprises a DNA sequence having at least
one variation in SEQ ID NO:1, wherein the at least one variation
comprises:
[0355] a G to A transition in the third position of codon 83;
[0356] a C to T transition in the first position of codon 101;
[0357] a G to A transition in the second position of codon 101;
[0358] a C to T transition in the first position of codon 247;
[0359] a C to T transition in the third position of codon 342;
[0360] a C to T transition in the third position of codon 421;
[0361] an A to G transition in the second position of codon
437;
[0362] a T to C transition in the first position of codon 497;
or
[0363] a combination thereof along with degenerate variants
thereof, fragments thereof, or analogs or derivatives thereof;
[0364] f) an isolated nucleic acid molecule hybridizable under
standard hybridization conditions to an isolated variant allele of
the PRODH gene, degenerate variants thereof, fragments thereof, or
analogs or derivatives thereof;
[0365] g) an isolated variant of a murine Prodh gene comprising a
DNA sequence of SEQ ID NO:7, degenerate variants thereof, fragments
thereof, or analogs or derivatives thereof;
[0366] h) an isolated nucleic acid molecule hybridizable under
standard hybridization conditions to an isolated nucleic acid
molecule comprising the DNA sequence of SEQ ID NO:7, degenerate
variants thereof, fragments thereof, or analogs or derivatives
thereof;
[0367] i) an isolated nucleic acid molecule comprising a DNA
sequence of SEQ ID NO:9, degenerate variants thereof, fragments
thereof, or analogs or derivatives thereof; and
[0368] j) an isolated nucleic acid molecule hybridizable under
standard hybridization conditions to an isolated nucleic acid
molecule comprising a DNA sequence of SEQ ID NO:9, degenerate
variants thereof, fragments thereof, or analogs or derivatives
thereof.
[0369] As used herein, an "isolated protein of the invention"
refers to any of the following:
[0370] a) an isolated protein comprising an amino acid sequence of
SEQ ID NO:2, conservative variants thereof, fragments thereof, or
analogs or derivatives thereof;
[0371] b) an isolated protein comprising an amino sequence of SEQ
ID NO:4, conservative variants thereof, fragments thereof, or
analogs or derivatives thereof; and
[0372] c) an isolated protein comprising an amino acid sequence
having at least one variation in SEQ ID NO:2, wherein the at least
one variation comprises:
[0373] Arg101Trp;
[0374] Arg101Glu;
[0375] Glu437Arg; or
[0376] a combination thereof;
[0377] d) an isolated mutant variant proline dehydrogenase (Prodh)
comprising an amino acid sequence of SEQ ID NO:8, conservative
variants thereof, fragments thereof, or analogs or derivatives
thereof.
[0378] Degenerate Variants of an Isolated Nucleic Acid Molecule of
the Invention, and Conservative Variants of an Isolated Protein of
the Invention
[0379] Due to the degenerate nature of codons in the genetic code,
an isolated protein of the invention can be encoded by nucleic acid
molecules other than an isolated nucleic acid molecule of the
invention. "Degenerate nature" refers to the use of different
three-letter codons to specify a particular amino acid pursuant to
the genetic code. It is well known in the art that the following
codons can be used interchangeably to code for each specific amino
acid:
[0380] Phenylalanine (Phe or F)
[0381] UUU or UUC
[0382] Leucine (Leu or L)
[0383] UUA or UUG or CUU or CUC or CUA or CUG
[0384] Isoleucine (IIe or I)
[0385] AUU or AUC or AUA
[0386] Methionine (Met or M)
[0387] AUG
[0388] Valine (Val or V)
[0389] GUU or GUC of GUA or GUG
[0390] Serine (Ser or S)
[0391] UCU or UCC or UCA or UCG or AGU or AGC
[0392] Proline (Pro or P)
[0393] CCU or CCC or CCA or CCG
[0394] Threonine (Thr or T)
[0395] ACU or ACC or ACA or ACG
[0396] Alanine (Ala or A)
[0397] GCU or GCG or GCA or GCG
[0398] Tyrosine (Tyr or Y)
[0399] UAU or UAC
[0400] Histidine (His or H)
[0401] CAU or CAC
[0402] Glutamine (Gln or Q)
[0403] CAA or CAG
[0404] Asparagine (Asn or N)
[0405] AAU or AAC
[0406] Lysine (Lys or K)
[0407] AAA or AAG
[0408] Aspartic Acid (Asp or D)
[0409] GAU or GAC
[0410] Glutamic Acid (Glu or E)
[0411] GAA or GAG
[0412] Cysteine (Cys or C)
[0413] UGU or UGC
[0414] Arginine (Arg or R)
[0415] CGU or CGC or CGA or CGG or AGA or AGG
[0416] Glycine (Gly or G)
[0417] GGU or GGC or GGA or GGG
[0418] Tryptophan (Trp or W)
[0419] UGG
[0420] Termination codon
[0421] UAA (ochre) or UAG (amber) or UGA (opal)
[0422] It should be understood that the codons specified above are
for RNA sequences. The corresponding codons for DNA have a T
substituted for U.
[0423] Likewise, conservative isolated variants of an PRODH protein
of the present invention include, but are not limited to, those
containing, as a primary amino acid sequence, substitutions of
amino acid residues in the amino acid sequences of SEQ ID NO:2, as
well as in a variant PRODH protein of the invention having an amino
acid sequence as described above, a murine Prodh comprising an
amino acid sequence of SEQ ID NO:4, and a mutant murine Prodh
comprising an amino acid sequence of SEQ ID NO:8, wherein the
substituted amino acids are functionally equivalent to the amino
acid residues for which they substitute. For example, one or more
amino acid residues within the sequence can be substituted by
another amino acid of a similar polarity, which acts as a
functional equivalent, resulting in a silent alteration.
Substitutes for an amino acid within the sequence may be selected
from other members of the class to which the amino acid residue
belongs. For example, the nonpolar (hydrophobic) amino acids
include alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan and methionine. Amino acids containing
aromatic ring structures are phenylalanine, tryptophan, and
tyrosine. The polar neutral amino acids include glycine, serine,
threonine, cysteine, tyrosine, asparagine, and glutamine. The
positively charged (basic) amino acids include arginine, lysine and
histidine. The negatively charged (acidic) amino acids include
aspartic acid and glutamic acid. Such alterations will not be
expected to affect apparent molecular weight as determined by
polyacrylamide gel electrophoresis, or isoelectric point.
[0424] Particularly preferred substitutions are:
[0425] Lys for Arg and vice versa such that a positive charge may
be maintained;
[0426] Glu for Asp and vice versa such that a negative charge may
be maintained;
[0427] Ser for Thr such that a free --OH can be maintained; and
[0428] Gln for Asn such that a free NH.sub.2 can be maintained.
[0429] Amino acid substitutions may also be introduced to
substitute an amino acid with a particularly preferable property.
For example, a Cys may be introduced at a potential site for
disulfide bridges with another Cys. A His may be introduced as a
particularly "catalytic" site (i.e., His can act as an acid or base
and is the most common amino acid in biochemical catalysis). Pro
may be introduced because of its particularly planar structure,
which induces .beta.-turns in the protein's structure. As a result
of such substitutions, "conservative variants" of an amino acid
sequence of the invention are formed.
[0430] Fragments of Isolated Nucleic Acid Molecules and Isolated
Proteins of the Invention
[0431] Further, as used herein, a "fragment" of an isolated nucleic
acid molecule of the invention is defined as an isolated nucleic
acid molecule comprising at least 10 contiguous nucleotides,
particularly at least 20 contiguous nucleotides, more particularly
at least 30 contiguous nucleotides, and even more particularly at
least 40 contiguous nucleotides of an isolated nucleic acid
molecule of the invention in the same 5'-3' order the contiguous
nucleotides appear in the isolated nucleic acid molecule of the
invention. Fragments of an isolated nucleic acid molecule of the
invention can readily be prepared by digesting an isolated nucleic
acid molecule of the invention with a restriction endonuclease.
Alternatively, one may use DNAse in the presence of manganese to
fragment the DNA, or the DNA can be physically sheared, as for
example, by sonication. The DNA fragments of an isolated nucleic
acid molecule of the invention can then be separated according to
size by standard techniques, including but not limited to, agarose
and polyacrylamide gel electrophoresis and column chromatography.
Once fragments of the isolated nucleic acid molecule have
themselves been separated, they can be readily inserted the
fragments into expression vectors. As a result, the peptides and
polypeptides, which are themselves fragments of an isolated protein
of the invention can readily be produced by one of ordinary skill
in the art.
[0432] Likewise, a "fragment" of protein of the invention comprises
at least 10 contiguous amino acid residues, particularly at least
15 contiguous amino acid residues, even more particularly at least
20 contiguous amino acid residues, and most particularly at least
25 contiguous amino acid residues of a protein of the invention, in
the N terminus to C terminus order in which the contiguous residues
occur in the protein of the invention. One of ordinary skill in the
art can readily prepare such fragments using recombinant DNA
techniques with fragments of isolated nucleic acid molecules of the
invention, by digesting a protein of the invention with a protease,
chemically cleaving a protein of the invention with chemical
reagents such as CNBr, or synthesizing the fragment using routine
solid support methods as taught by Merrifield.
[0433] As used herein, the term "sequence homology" in all its
grammatical forms refers to the relationship between proteins that
possess a "common evolutionary origin," including proteins from
superfamilies (e.g., the immunoglobulin superfamily) and homologous
proteins from different species (e.g., myosin light chain, etc.)
(Reeck et al., 1987, Cell 50:667).
[0434] Accordingly, the term "sequence similarity" in all its
grammatical forms refers to the degree of identity or
correspondence between nucleic acid or amino acid sequences of
proteins that do not share a common evolutionary origin (see Reeck
et al., supra). However, in common usage and in the instant
application, the term "homologous," when modified with an adverb
such as "highly," may refer to sequence similarity and not a common
evolutionary origin.
[0435] In a specific embodiment, two DNA sequences are
"substantially homologous" or "substantially similar" when at least
about 50% (preferably at least about 75%, and most preferably at
least about 90 or 95%) of the nucleotides match over the defined
length of the DNA sequences. Sequences that are substantially
homologous can be identified by comparing the sequences using
standard software available in sequence data banks, or in a
Southern hybridization experiment under, for example, stringent
conditions as defined for that particular system. Defining
appropriate hybridization conditions is within the skill of the
art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I &
II, supra; Nucleic Acid Hybridization, supra.
[0436] Similarly, in a particular embodiment, two amino acid
sequences are "substantially homologous" or "substantially similar"
when greater than 30% of the amino acids are identical, or greater
than about 60% are similar (functionally identical). Preferably,
the similar or homologous sequences are identified by alignment
using, for example, the GCG (Genetics Computer Group, Program
Manual for the GCG Package, Version 7, Madison, Wis.) pileup
program-using default parameters.
[0437] The term "corresponding to" is used herein to refer to
similar or homologous sequences, whether the exact position is
identical or different from the molecule to which the similarity or
homology is measured. Thus, the term "corresponding to" refers to
the sequence similarity, and not the numbering of the amino acid
residues or nucleotide bases.
[0438] A variant allele of the human PRODH gene, whether genomic
DNA or cDNA, can be isolated from any source, particularly from a
human cDNA or genomic library. Likewise, a variant allele of Prodh,
whether genomic or cDNA can be isolated from any source,
particularly from a murine cDNA or genomic library. Methods for
obtaining a variant allele of a PRODH gene or a Prodh gene are well
known in the art, as described above (see, e.g., Sambrook et al.,
1989, supra).
[0439] The DNA may be obtained by standard procedures known in the
art from cloned DNA (e.g., a DNA "library"), and preferably is
obtained from a cDNA library prepared from tissues with high level
expression of a PRODH protein, an isolated variant thereof, or
Prodh or an isolated variant thereof, by chemical synthesis, by
cDNA cloning, or by the cloning of genomic DNA, or fragments
thereof, purified from the desired cell (See, for example, Sambrook
et al., 1989, supra; Glover, D. M. (ed.), 1985, DNA Cloning: A
Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. 1, II).
Clones derived from genomic DNA may contain regulatory and intron
DNA regions in addition to coding regions; clones derived from cDNA
will not contain intron sequences. Whatever the source, an isolated
nucleic acid molecule of the invention can be molecularly cloned
into a suitable vector for propagation.
[0440] Once the DNA fragments are generated, identification of a
specific DNA fragment comprising an isolated nucleic acid molecule
of the invention may be accomplished in a number of ways. For
example, if an amount of a portion of an isolated nucleic acid
molecule is available and can be purified and labeled, the
generated DNA fragments may be screened by nucleic acid
hybridization to a labeled probe (Benton and Davis, 1977, Science
196:180; Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A.
72:3961). For example, a set of oligonucleotides corresponding to
the partial amino acid sequence information obtained SEQ ID NO: 2
or a variant human PRODH protein, SEQ ID NO:4, or SEQ ID NO:8 of
the present invention can be prepared and used as probes for an
isolated nucleic acid molecule of the invention, as was done in a
specific example, infra, or as primers for cDNA or mRNA (e.g., in
combination with a poly-T primer for RT-PCR). Preferably, a
fragment is selected that is highly unique to an isolated nucleic
acid molecule of the invention. Those DNA fragments with
substantial homology to the probe will hybridize. As noted above,
the greater the degree of homology, the more stringent
hybridization conditions can be used.
[0441] Further selection can be carried out on the basis of the
properties the protein produced from expression of an isolated
nucleic acid molecule of the invention. For example, an isolated
variant allele of a PRODH gene of the present invention can be
isolated if it encodes a variant PRODH protein having an
isoelectric, electrophoretic, amino acid composition, or partial
amino acid sequence different from PRODH produced from the
expression of the PRODH gene (SEQ ID NO:1) herein. Thus, the
presence of a variant allele of a PRODH gene of the present
invention may be detected by assays based on the physical,
chemical, or immunological properties of its expressed product. For
example, cDNA clones, or DNA clones which hybrid-select the proper
mRNAs, can be selected which produce a protein that, e.g., has
different electrophoretic migration, isoelectric focusing or
non-equilibrium pH gel electrophoresis behavior, proteolytic
digestion maps, or antigenic properties as are known for a PRODH
protein comprising an amino acid sequence of SEQ ID NO:2. Such
selection can also be made between Prodh and an isolated variant of
Prodh.
[0442] An isolated variant allele of a PRODH gene of the present
invention, for example, can also be identified by mRNA selection,
i.e., by nucleic acid hybridization followed by in vitro
translation. In this procedure, nucleotide fragments are used to
isolate complementary mRNAs by hybridization. Such DNA fragments
may represent available, purified DNA of an isolated variant allele
of a human PRODH gene of the present invention, or may be synthetic
oligonucleotides designed from the partial amino acid sequence
information.
[0443] Immunoprecipitation analysis or functional assays of the in
vitro translation products of the products of the isolated mRNAs
identifies the mRNA and, therefore, the complementary DNA
fragments, that contain the desired sequences. Naturally, these
techniques can also be used to identify isolated variant of
Prodh.
[0444] Furthermore, a detectably labeled isolated nucleic acid
molecule of the present invention can be prepared by one of
ordinary skill in the art. Once detectably labeled, an isolated
nucleic acid molecule of the invention degenerate variants thereof,
fragments thereof, or analogs or derivatives thereof, can then be
used as a probe to identify homologous DNA fragments from among
other genomic DNA fragments. Suitable labels include enzymes,
radioactive isotopes, fluorophores (e.g., fluorescene
isothiocyanate (FITC), phycoerythrin (PE), Texas red (TR),
rhodamine, free or chelated lanthanide series salts, especially
Eu.sup.3+, to name a few fluorophores), chromophores,
radioisotopes, chelating agents, dyes, colloidal gold, latex
particles, ligands (e.g., biotin), and chemiluminescent agents.
When a control marker is employed, the same or different labels may
be used for the receptor and control marker.
[0445] In the instance where a radioactive label, such as the
isotopes .sup.3H, .sup.14C, .sup.32P, .sup.35S, .sup.36Cl,
.sup.51Cr, .sup.57Co, .sup.58Co, .sup.59Fe, .sup.90Y, .sup.125I,
.sup.131I and .sup.186Re are used, known currently available
counting procedures may be utilized. In the instance where the
label is an enzyme, detection may be accomplished by any of the
presently utilized colorimetric, spectrophotometric,
filuorospectrophotometric, amperometric or gasometric techniques
known in the art.
[0446] Direct labels are one example of labels which can be used
according to the present invention. A direct label has been defined
as an entity, which in its natural state, is readily visible,
either to the naked eye, or with the aid of an optical filter
and/or applied stimulation, e.g. U. V. light to promote
fluorescence. Examples of colored labels, which can be used
according to the present invention include metallic sol particles,
for example, gold sol particles such as those described by
Leuvering (U.S. Pat. No. 4,313,734); dye sol particles such as
described by Gribnau et al. (U.S. Pat. No. 4,373,932) and May et
al. (WO 88108534); dyed latex such as described by May, supra,
Snyder (EP-A 0 280 559 and 0 281 327); or dyes encapsulated in
liposomes as described by Campbell et al. (U.S. Pat. No.
4,703,017). Other direct labels include a radionucleotide, a
fluorescent moiety or a luminescent moiety. In addition to these
direct labeling devices, indirect labels comprising enzymes can
also be used according to the present invention. Various types of
enzyme linked immunoassays are well known in the art, for example,
alkaline phosphatase and horseradish peroxidase, lysozyme,
glucose-6-phosphate dehydrogenase, lactate dehydrogenase, and
urease. These and others have been discussed in detail by Eva
Engvall in Enzyme Immunoassay ELISA and EMIT in Methods in
Enzymology, 70. 419-439, 1980 and in U.S. Pat. No. 4,857,453.
[0447] Other labels for use in the invention include magnetic beads
or magnetic resonance imaging labels.
Cloning Vectors
[0448] As explained above, the present invention extends to various
cloning vectors. In particular, the present invention extends to a
cloning vector comprising an isolated nucleic acid molecule of the
invention and an origin of replication.
[0449] A large number of vector-host systems known in the art may
be used to clone an isolated nucleic acid molecule of the
invention. Possible vectors include, but are not limited to,
plasmids or modified viruses. The vector system used however must
be compatible with the host cell. Examples of vectors that are
commercially available and have applications herein include, but
are not limited to, E. coli, bacteriophages such as lambda
derivatives, or plasmids such as pBR322 derivatives or pUC plasmid
derivatives, e.g., pGEX vectors, pmal-c, pFLAG, etc. Furthermore,
insertion of an isolated nucleic acid molecule of the invention as
described above into a cloning vector can be readily accomplished
by one of ordinary skill in the art. For example, insertion can be
accomplished by ligating an isolated nucleic acid molecule of the
invention into a cloning vector which has complementary cohesive
termini. However, if the complementary restriction sites used to
fragment the isolated nucleic acid molecule of the invention, to
form a cassette are not present in the cloning vector, the ends of
the cassette may be enzymatically modified using procedures well
known to one of ordinary skill in the art. Once modified, the
cassette can be readily ligated into a commercially available
cloning vector. Alternatively, any site desired may be produced by
ligating nucleotide sequences (linkers) onto the DNA termini of the
cassette; these ligated linkers may comprise specific chemically
synthesized oligonucleotides encoding restriction endonuclease
recognition sequences.
[0450] Once an isolated nucleic acid molecule of the invention is
inserted into a vector, the vector can then be introduced into host
cells via transformation, transfection, infection, electroporation,
etc., so that many copies of the isolated nucleic acid molecule of
the invention can be generated. Preferably, the cloned isolated
variant allele or isolated nucleic acid molecule hybridizable
thereto under standard hybridization conditions is contained on a
shuttle vector plasmid, which provides for expansion in a cloning
cell, e.g., E. coli, and facile purification for subsequent
insertion into an appropriate expression cell line, if such is
desired. For example, a shuttle vector, which is a vector that can
replicate in more than one type of organism, can be prepared for
replication in both E. coli and Saccharomyces cerevisiae by linking
sequences from an E. coli plasmid with sequences from the yeast 2
.mu.plasmid.
[0451] In an alternative method, an isolated nucleic acid molecule
of the invention may be identified and isolated after insertion
into a suitable cloning vector in a "shot gun" approach. Enrichment
for an isolated variant allele, for example, by size fractionation,
can be done before insertion into the cloning vector.
Expression Vectors
[0452] An isolated nucleic acid molecule of the inventioin can be
inserted into an appropriate expression vector, i.e., a vector
which contains the necessary elements for the transcription and
translation of the inserted protein-coding sequence such that the
protein-coding sequence is operatively associated with a promoter.
A DNA sequence is "operatively associated" to an expression control
sequence, such as a promoter, when the expression control sequence
controls and regulates the transcription and translation of that
DNA sequence. The term "operatively associated" includes having an
appropriate start signal (e.g., ATG) in front of the DNA sequence
to be expressed and maintaining the correct reading frame to permit
expression of the DNA sequence under the control of the expression
control sequence and production of the desired product encoded by
the DNA sequence. If an isolated nucleic acid molecule of the
invention does not contain an appropriate start signal, such a
start signal can be readily inserted into the expression vector in
front of (5' of) protein encoding nucleic acid molecule inserted
into the expression vector using methods readily understood and
available to one of ordinary skill in the art.
[0453] Both cDNA and genomic sequences can be cloned and expressed
under control of such regulatory sequences. An expression vector
also preferably includes a replication origin.
[0454] The necessary transcriptional and translational signals can
be provided on a recombinant expression vector, or they may be
supplied by an isolated nucleic acid molecule of the invention
inserted into an expression vector.
[0455] Potential host-vector systems which are commercially
available and have ready applications herein include, but are not
limited to mammalian cell systems infected with virus (e.g.,
vaccinia virus, adenovirus, etc.); insect cell systems infected
with virus (e.g., baculovirus); microorganisms such as yeast
containing yeast vectors; or bacteria transformed with
bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression
elements of vectors vary in their strengths and specificities.
Depending on the host-vector system utilized, any one of a number
of suitable transcription and translation elements may be used.
[0456] An isolated nucleic acid molecule of the invention may be
expressed chromosomally, after integration of the coding sequence
by recombination. In this regard, any of a number of amplification
systems may be used to achieve high levels of stable gene
expression (See Sambrook et al., 1989, supra).
[0457] A unicellular host transformed or transfected with an
expression vector of the invention can be cultured in an
appropriate cell culture medium that provides for expression of an
isolated nucleic acid molecule of the invention inserted into an
expression vector and operatively associated with a promoter.
[0458] Any of the methods previously described for the insertion of
DNA fragments into a cloning vector may be used to construct
expression vectors of the present invention. These methods may
include in vitro recombinant DNA and synthetic techniques and in
vivo recombination (genetic recombination).
[0459] Expression of an isolated nucleic acid molecule of the
invention to produce a protein of the invention may be controlled
by any promoter/enhancer element known in the art. However, these
regulatory elements must be functional in the host selected for
expression. Promoters which may be used to control expression
include, but are not limited to, the SV40 early promoter region
(Benoist and Chambon, 1981, Nature 290:304-310), the promoter
contained in the 3' long terminal repeat of Rous sarcoma virus
(Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine
kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.
78:1441-1445), the regulatory sequences of the metallothionein gene
(Brinster et al., 1982, Nature 296:39-42); prokaryotic expression
vectors such as the .beta.-lactamase promoter (Villa-Kamaroff, et
al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac
promoter (DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A.
80:21-25); see also "Useful proteins from recombinant bacteria" in
Scientific American, 1980, 242:74-94; promoter elements from yeast
or other fungi such as the Gal 4 promoter, the ADC (alcohol
dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter,
alkaline phosphatase promoter; and the animal transcriptional
control regions, which exhibit tissue specificity and have been
utilized in transgenic animals: elastase I gene control region
which is active in pancreatic acinar cells (Swift et al., 1984,
Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp.
Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515);
insulin gene control region which is active in pancreatic beta
cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin gene
control region which is active in lymphoid cells (Grosschedl et
al., 1984, Cell 38:647-658; Adames et al., 1985, Nature
318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444),
mouse mammary tumor virus control region which is active in
testicular, breast, lymphoid and mast cells (Leder et al., 1986,
Cell 45:485-495), albumin gene control region which is active in
liver (Pinkert et al., 1987, Genes and Devel. 1:268-276),
alpha-fetoprotein gene control region which is active in liver
(Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et
al., 1987, Science 235:53-58), alpha 1-antitrypsin gene control
region which is active in the liver (Kelsey et al., 1987, Genes and
Devel. 1:161-171), beta-globin gene control region which is active
in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias
et al., 1986, Cell 46:89-94), myelin basic protein gene control
region which is active in oligodendrocyte cells in the brain
(Readhead et al., 1987, Cell 48:703-712), myosin light chain-2 gene
control region which is active in skeletal muscle (Sani, 1985,
Nature 314:283-286), and gonadotropic releasing hormone gene
control region which is active in the hypothalamus (Mason et al.,
1986, Science 234:1372-1378).
[0460] Moreover, an expression vector can be identified by four
general approaches: (a) PCR amplification of the desired plasmid
DNA or specific mRNA, (b) nucleic acid hybridization, (c) presence
or absence of selection marker gene functions, and (d) expression
of inserted sequences. In the first approach, an isolated nucleic
acid molecule of the invention can be amplified by PCR to provide
for detection of the amplified product. In the second approach, the
presence of a foreign gene inserted in an expression vector of the
present invention can be detected by nucleic acid hybridization
using probes comprising sequences that are homologous to an
inserted marker gene. In the third approach, the recombinant
vector/host system can be identified and selected based upon the
presence or absence of certain "selection marker" gene functions
(e.g., .beta.-galactosidase activity, thymidine kinase activity,
resistance to antibiotics, transformation phenotype, occlusion body
formation in baculovirus, etc.) caused by the insertion of foreign
genes in the vector. In yet another example, if, an isolated
nucleic acid molecule of the invention is inserted within the
"selection marker" gene sequence of the vector, recombinants
containing the insert can be identified by the absence of the
inserted gene function. In the fourth approach, recombinant
expression vectors can be identified by assaying for the activity,
biochemical, or immunological characteristics of the gene product
expressed by the recombinant expression vector, provided that the
expressed protein assumes a functionally active conformation.
[0461] Naturally, the present invention extends to a method of
producing an isolated protein of the invention. An example of such
a method comprises the steps of culturing a unicellular host
transformed or transfected with an expression vector of the
invention under conditions that provide for expression of the
isolated nucleic acid molecule of the invention inserted into the
expression vector, to produce a protein of the invention. The
protein produced depends upon which isolated nucleic acid molecule
of the invention was inserted into the expression vector. The
isolated protein produced can then be readily recovered from the
unicellular host, the culture, or both.
[0462] A wide variety of unicellular host/expression vector
combinations commercially available to the skilled artisan may be
employed in producing an isolated protein of the invention.
[0463] Useful expression vectors, for example, may consist of
segments of chromosomal, non-chromosomal and synthetic DNA
sequences. Suitable vectors include derivatives of SV40 and known
bacterial plasmids, e.g., E. coli plasmids col E1, pCR1, pBR322,
pMa1-C2, pET, pGEX (Smith et-al, 1988, Gene 67:31-40), pMB9 and
their derivatives, plasmids such as RP4; phage DNAS, e.g., the
numerous derivatives of phage .lambda., e.g., NM989, and other
phage DNA, e.g., M13 and filamentous single stranded phage DNA;
yeast plasmids such as the 2.mu. plasmid or derivatives thereof;
vectors useful in eukaryotic cells, such as vectors useful in
insect or mammalian cells; vectors derived from combinations of
plasmids and phage DNAs, such as plasmids that have been modified
to employ phage DNA or other expression control sequences; and the
like.
[0464] For example, in baculovirus expression systems, both
non-fusion transfer vectors, such as but not limited to pVL941
(BamH1 cloning site; Summers), pVL1393 (BamH1, SmaI, XbaI, EcoR1,
NotI, XmaIII, BglII, and PstI cloning site; Invitrogen), pVL1392
(BglII, PstI, NotI, XmaIII, EcoR1, XbaI, SmaI, and BamH1 cloning
site; Summers and Invitrogen), and pBlueBacIII (BamH1, BglII, PstI,
NcoI, and HindIII cloning site, with blue/white recombinant
screening possible; Invitrogen), and fusion transfer vectors, such
as but not limited to pAc700 (BamH1 and KpnI cloning site, in which
the BamH1 recognition site begins with the initiation codon;
Summers), pAc70l and pAc702 (same as pAc700, with different reading
frames), pAc360 (BamH1 cloning site 36 base pairs downstream of a
polyhedrin initiation codon; Invitrogen(195)), and pBlueBacHisA, B,
C (three different reading frames, with BamH1, BglII, PstI, NcoI,
and HindIII cloning site, an N-terminal peptide for ProBond
purification, and blue/white recombinant screening of plaques;
Invitrogen (220) can be used.
[0465] Mammalian expression vectors contemplated for use in the
invention include vectors with inducible promoters, such as the
dihydrofolate reductase (DHFR) promoter, e.g., any expression
vector with a DHFR expression vector, or a DHFR/methotrexate
co-amplification vector, such as pED PstI, SalI, SbaI, SmaI, and
EcoRI cloning site, with the vector expressing both the cloned gene
and DHFR; see Kaufman, Current Protocols in Molecular Biology,
16.12(1991).
[0466] Alternatively, a glutamine synthetase/methionine sulfoximine
co-amplification vector, such as pEE14 (HindIII, XbaI, SmaI, SbaI,
EcoRI, and BclI cloning site, in which the vector expresses
glutamine synthase and the cloned gene; Celltech). In another
embodiment, a vector that directs episomal expression under control
of Epstein Barr Virus (EBV) can be used, such as pREP4 (BamH1,
SfiI, XhoI, NotI, NheI, HindIII, AheI, PvuII, and KpnI cloning
site, constitutive RSV-LTR promoter, hygromycin selectable marker;
Invitrogen), pCEP4 (BamH1, SfiI, XhoI, NotI, NheI, HindIII, NheI,
PvuII, and KpnI cloning site, constitutive hCMV immediate early
gene, hygromycin selectable marker; Invitrogen), pMEP4 (KpnI, PvuI,
NheI, HindIII, NotI, XhoI, SfiI, BamH1 cloning site, inducible
metallothionein IIa gene promoter, hygromycin selectable marker:
Invitrogen), pREP8 (BamH1, XhoI, NotI, HindIII, NheI, and KpnI
cloning site, RSV-LTR promoter, histidinol selectable marker;
Invitrogen), pREP9 (KpnI, NheI, HindIII, NotI, XhoI, SfiI, and
BamHI cloning site, RSV-LTR promoter, G418 selectable marker;
Invitrogen), and pEBVHis (RSV-LTR promoter, hygromycin selectable
marker, N-terminal peptide purifiable via ProBond resin and cleaved
by enterokinase; Invitrogen). Selectable mammalian expression
vectors for use in the invention include pRc/CMV (HindIII, BstXI,
NotI, SbaI, and ApaI cloning site, G418 selection; Invitrogen),
pRc/RSV (HindIII, SpeI, BstXI, NotI, XbaI cloning site, G418
selection; Invitrogen), and others. Vaccinia virus mammalian
expression vectors (see, Kaufman, 1991, supra) for use according to
the invention include but are not limited to pSC11 (SmaI cloning
site, TK- and .beta.-gal selection), pMJ601 (SalI, SmaI, AflI,
NarI, BspMII, BamHI, ApaI, NheI, SaclI, KpnI, and HindIII cloning
site; TK- and .beta.-gal selection), and pTKgptF1S (EcoR1, PstI,
SalI, AccI, HindIII, SbaI, BamHI, and Hpa cloning site, TK or XPRT
selection).
[0467] Yeast expression systems can also be used according to the
invention include the non-fusion pYES2 vector (XbaI, SphI, ShoI,
NotI, GstXI, EcoRI, BstXI, BamHI, Saci, KpnI, and HindIII cloning
sit; Invitrogen) or the fusion pYESHisA, B, C (XbaI, SphI, ShoI,
NotI, BstXI, EcoR1, BamH 1, SacI, KpnI, and HindIII cloning site,
N-terminal peptide purified with ProBond resin and cleaved with
enterokinase; Invitrogen), to mention just two, can be employed
according to the invention.
[0468] Once a particular recombinant DNA molecule is identified and
isolated, several methods known in the art may be used to propagate
it. Furthermore, once a suitable host system and growth conditions
are established, recombinant expression vectors can be propagated
and prepared in quantity. As previously explained, the expression
vectors which can be used include, but are not limited to, the
following vectors or their derivatives: human or animal viruses
such as vaccinia virus or adenovirus; insect viruses such as
baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda),
and plasmid and cosmid DNA vectors, to name but a few.
[0469] Examples of unicellular hosts contemplated by the present
invention, which are well know to those of ordinary skill in the
art, include but are not limited to, E. coli Pseudonomas, Bacillus,
Strepomyces, yeast, CHO, R1.1, B-W, L-M, COS1, COS7, BSC1, BSC40,
BMT10, or HeLa cells, all of which are readily available to the
skilled artisan. In addition, a host cell strain may be chosen
which modulates the expression of an isolated nucleic acid molecule
of the invention modifies and processes the gene product in the
specific fashion desired. Different host cells have characteristic
and specific mechanisms for the translational and
post-translational processing and modification (e.g.,
glycosylation, cleavage [e.g., of signal sequence]) of proteins.
Appropriate cell lines or host systems can be chosen to ensure the
desired modification and processing of the foreign protein
expressed. For example, expression in a bacterial system can be
used to produce an nonglycosylated core protein product. However, a
translocation signal sequence of the product produced from
expression of an isolated nucleic acid molecule of the invention in
bacteria may not be properly spliced. Expression in yeast can
produce a glycosylated product. Expression in eukaryotic cells can
increase the likelihood of "native" glycosylation and folding.
Moreover, expression in mammalian cells can provide a tool for
reconstituting, or constituting activity of murine proline
dehydrogenase, human PRODH, or a variant human PRODH protein of the
invention. Furthermore, different vector/host expression systems
may affect processing reactions, such as proteolytic cleavages, to
a different extent.
[0470] Vectors are introduced into the desired unicellular hosts by
methods known in the art, e.g., transfection, electroporation,
microinjection, transduction, cell fusion, DEAE dextran, calcium
phosphate precipitation, lipofection (lysosome fusion), use of a
gene gun, or a DNA vector transporter (see, e.g., Wu et al., 1992,
J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem.
263:14621-14624; Hartmut et al., Canadian Patent Application No.
2,012,311, filed Mar. 15, 1990).
Antibodies to an Isolated Protein of the Invention
[0471] According to the invention, an isolated protein of the
invention may be used as an immunogen to generate antibodies. Such
antibodies include but are not limited to polyclonal, monoclonal,
chimeric, single chain, Fab fragments, and an Fab expression
library. Furthermore, antibodies of the invention may be cross
reactive, e.g., they may recognize more than one isolated protein
of the invention.
[0472] Various procedures known in the art may be used for the
production of polyclonal antibodies of the invention. For the
production of antibody, various host animals can be immunized by
injection with an immunogen described above. Examples of such
animals include, but are not limited to rabbits, mice, rats, sheep,
goats, etc. In one embodiment, an isolated protein of the invention
can be conjugated to an immunogenic carrier, e.g., bovine serum
albumin (BSA) or keyhole limpet hemocyanin (KLH). Various adjuvants
may be used to increase the immunological response, depending on
the host species, including but not limited to Freund's (complete
and incomplete), mineral gels such as aluminum hydroxide, surface
active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and potentially useful human adjuvants such as BCG
(bacille Calmette-Guerin) and Corynebacterium parvum.
[0473] For preparation of monoclonal antibodies of the invention,
any technique that provides for the production of antibody
molecules by continuous cell lines in culture may be used. These
include but are not limited to the hybridoma technique originally
developed by Kohler and Milstein [Nature 256:495-497 (1975)], as
well as the trioma technique, the human B-cell hybridoma technique
[Kozbor et al., Immunology Today 4:72 1983); Cote et al., Proc.
Natl. Acad. Sci. U.S.A. 80:2026-2030 (1983)], and the EBV-hybridoma
technique to produce human monoclonal antibodies [Cole et al., in
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.
77-96 (1985)]. In an additional embodiment of the invention,
monoclonal antibodies can be produced in germ-free animals
utilizing technology set forth in PCT/US90/02545. In fact,
according to the invention, techniques developed for the production
of "chimeric antibodies" [Morrison et al., J. Bacteriol. 159:870
(1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al.,
Nature 314:452-454 (1985)] by splicing the genes from a mouse which
encode an antibody molecule specific for a protein of the invention
together with genes from a human antibody molecule of appropriate
biological activity can be used, and are within the scope of this
invention. Such human or humanized chimeric antibodies are
preferred for use in determining the presence of an isolated
protein of the invention in a sample.
[0474] According to the invention, techniques described for the
production of single chain antibodies [U.S. Pat. Nos. 5,476,786 and
5,132,405 to Huston; U.S. Pat. No. 4,946,778] can be adapted to
produce particular isolated variant human PRODH protein-specific
single chain antibodies. An additional embodiment of the invention
utilizes the techniques described for the construction of Fab
expression libraries [Huse et al., Science 246:1275-1281 (1989)] to
allow rapid and easy identification of monoclonal Fab fragments
with the desired specificity for an isolated protein of the
invention.
[0475] Antibody fragments which contain the idiotype of the
antibody molecule can be generated by known techniques. For
example, such fragments include but are not limited to: the
F(ab').sub.2 fragment which can be produced by pepsin digestion of
the antibody molecule; the Fab' fragments which can be generated by
reducing the disulfide bridges of the F(ab').sub.2 fragment, and
the Fab fragments which can be generated by treating the antibody
molecule with papain and a reducing agent.
[0476] In the production of antibodies, screening for the desired
antibody can be accomplished by techniques known in the art, e.g.,
radioimmunoassay, ELISA (enzyme-linked immunosorbant assay),
"sandwich" immunoassays, immunoradiometric assays, gel diffusion
precipitin reactions, immunodiffusion assays, in situ immunoassays
(using colloidal gold, enzyme or radioisotope labels, for example),
western blots, precipitation reactions, agglutination assays (e.g.,
gel agglutination assays, hemagglutination assays), complement
fixation assays, immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc. In one embodiment, antibody
binding is detected by detecting a label on the primary antibody.
In another embodiment, the primary antibody is detected by
detecting binding of a secondary antibody or reagent to the primary
antibody. In a further embodiment, the secondary antibody is
labeled. Many means are known in the art for detecting binding in
an immunoassay and are within the scope of the present invention.
For example, to select antibodies which recognize a specific
epitope of an isolated protein of the invention, one may assay
generated hybridomas for a product which binds to a fragment of a
variant PRODH protein containing such epitope.
[0477] The foregoing antibodies can be used in methods known in the
art relating to the localization and activity of an isolated
variant human PRODH protein, conservative variants thereof, or
fragments thereof e.g., for Western blotting, imaging a variant
human PRODH protein in situ, measuring levels thereof in
appropriate physiological samples, etc. using any of the detection
techniques mentioned above or known in the art.
Commercial Kits
[0478] In a further embodiment, the present invention extends to
commercial test kits suitable for use by a medical professional to
determine the presence or absence of predetermined PRODH activity,
or predetermined PRODH capability in target patient
populations.
[0479] In accordance with the testing techniques discussed above,
one class of such kits comprise at least the labeled PRODH or its
binding partner, for instance an antibody specific thereto, and
directions, of course, depending upon the method selected, e.g.,
"competitive," "sandwich," "DASP" and the like. The kits may also
contain peripheral reagents such as buffers, stabilizers, etc. In a
particular embodiment, the labeled PRODH comprises an amino acid
sequence of SEQ ID NO:2.
[0480] Another class of such kits may also include PCR reagents,
such as oligonucleotide primers, enzymes, gel matrixes, buffers,
etc.
[0481] Accordingly, a test kit may be prepared for the diagnosis or
detection of a susceptibility to schizophrenia, or a disease or
disorder related thereto, to measure levels of PRODH activity in a
bodily sample from a subject, wherein the kit comprises:
[0482] (a) a predetermined amount of at least one labeled
immunochemically reactive component obtained by the direct or
indirect attachment of the present PRODH factor or a specific
binding partner thereto, to a detectable label;
[0483] (b) other reagents; and
[0484] (c) directions, including comparison levels of PRODH, for
use of the kit.
[0485] An alternate kit for measuring the levels of PRODH activity
may comprise PCR reagents, such as oligonucleotide primers,
enzymes, gel matrices, buffers, directions, including comparison
levels of PRODH, for use of the kit. A still further alternate can
utilize reagents for measuring the levels of PRODH activity and
directions, including comparison levels of PRODH for use of the
kit.
[0486] In a further variation, a test kit may be prepared and used
for the purposes stated above, which operates according to a
predetermined protocol (e.g. "competitive," "sandwich," "double
antibody," etc.), and comprises:
[0487] (a) a labeled component which has been obtained by coupling
PRODH comprising an amino acid sequence of SEQ ID NO:2, a
conservative variant thereof, or fragment thereof to a detectable
label;
[0488] (b) one or more additional immunochemical reagents of which
at least one reagent is a ligand or an immobilized ligand, which
ligand is selected from the group consisting of:
[0489] (i) a ligand capable of binding with the labeled component
(a);
[0490] (ii) a ligand capable of binding with a binding partner of
the labeled component (a);
[0491] (iii) a ligand capable of binding with at least one of the
component(s) to be determined; and
[0492] (iv) a ligand capable of binding with at least one of the
binding partners of at least one of the component(s) to be
determined; and
[0493] (c) directions for the performance of a protocol for the
detection and/or determination of one or more components of an
immunochemical reaction between PRODH and a specific binding
partner thereto.
[0494] Other kits of the present invention utilize the discovery
that, unexpectedly, the presence of a particular variant allele of
the PRODH gene in the genome of a subject is indicative of an
increased susceptibility to schizophrenia or a disease or disorder
related thereto, or a diagnosis of schizophrenia in the subject.
Hence, the present invention extends to a test kit to facilitate
diagnosis and treatment of schizophrenia or a disease or disorder
related thereto in a eukaryotic cellular sample, wherein the test
kit comprises:
[0495] a) PCR oligonucleotide primers suitable for detection of an
isolated variant allele of a PRODH gene, wherein the PRODH gene
comprises a DNA sequence of SEQ ID NO:1, and the variant allele
comprises a DNA sequence having a variation in SEQ ID NO:1
comprising a transition of A to G in the second position of codon
437 of SEQ ID NO:1;
[0496] (b) other reagents such as enzymes, gel matrices, buffers,
etc.; and
[0497] (c) directions for use of the kit.
[0498] Assays for Screening the Ability Drugs and Therapeutic
Agents to Treat Schizophrenia or a Disease or Disorder Related
Thereto
[0499] In accordance with the above, an assay system for screening
potential drugs effective to modulate levels of human PRODH in a
subject may be prepared. The PRODH may be introduced into a test
system, such as a cell culture, and the prospective drug may also
be introduced into the cell culture. The cell culture is then
examined to observe any changes in the PRODH activity of the cells,
due either to the addition of the prospective drug alone, or due to
the effect of added quantities of the known PRODH.
[0500] Moreover, the present invention extends to an assay system
for screening drugs and other agents for their ability to treat
schizophrenia or a disease or disorder related thereto. Such an
assay of the present invention comprises the steps of culturing an
observable cellular test colony inoculated with the drug or agent
to be assayed, harvesting a cellular extract from the cellular test
colony, and examining the extract for the presence of PRODH. An
increase or decrease in the level of activity of PRODH in the test
colony compared to the level of activity of PRODH in a control
colony indicates the ability of the drug to modulate the
production, stability, degradation or activity of PRODH, which is
indicative of the ability of the drug or agent to treat
schizophrenia or a disease or disorder related thereto, such as
obsessive compulsive disorder (OCD), bipolar disorder (BP) or major
depressive disorder. An increase in the level of activity of PRODH
in the test colony compared to level of activity in a control
colony indicates the drug or agent is a potential therapeutic agent
for the treatment of schizophrenia or a disease or disorder related
thereto.
[0501] In another embodiment, the present invention extends to an
assay system for screening drugs, agents or compounds, to determine
their schizophrenic-related pharmacological activity. An example of
such a method comprises the steps of:
[0502] administering the compound to a mammal;
[0503] determining the level of activity of PRODH in the mammal;
and
[0504] comparing the level of activity of PRODH to the level of
activity of PRODH in a control animal to which the compound was not
administered. An increase in the level of activity of PRODH in the
mammal relative to the level of PRODH activity in the standard is
indicative that the drug, agent or compound may have
schizophrenic-related pharmacological activity, and ability as a
therapeutic agent for treating schizophrenia or a disease or
disorder related thereto, such as obsessive compulsive disorder
(OCD), bipolar disorder (BP) or major depressive disorder.
[0505] Another example of an assay system for screening drugs,
agents or compounds for schizophrenic related pharmacological
activity comprises the steps of:
[0506] determining a basal level of activity of PRODH in a
mammal;
[0507] administering the compound to the mammal;
[0508] determining the level of activity of PRODH in the mammal
after administration of the compound. An increase in the level of
activity of PRODH in the mammal relative to the basal level of
activity indicates the compound has a schizophrenic-related
pharmacological activity, and may have potential as a therapeutic
agent for treating schizophrenia or a disease or disorder related
thereto, such as obsessive compulsive disorder (OCD), bipolar
disorder (BP), or major depressive disorder (MDD).
[0509] Methods of assaying levels of activity of PRODH are readily
known to one of ordinary skill in the art, and are disclosed in
Blake, R. & Russel, E., Hyperprolinemia and prolinuria in a new
inbred strain of mice PRO/Re. Science 176, 809-811 (1972); Blake,
R. L., Animal Model for Hyperprolinaemia: Deficiency of Mouse
Proline Oxidase Activity Biochem. J. 129, 987-989 (1979) both of
which are hereby incorporated by reference in their entireties.
Treating Schizophrenia or a Disease or Disorder Related Thereto
[0510] In another embodiment, the present invention extends to a
method of treating schizophrenia or a disease or disorder related
thereto, such as obsessive compulsive disorder (OCD), bipolar
disorder (BP), or major depressive disorder (MDD) in a subject, or
a symptom of such a disease or disorder. An example of such a
method comprises administering to the subject a therapeutically
effective amount of isolated PRODH comprising an amino acid
sequence of SEQ ID NO:2, a conservative variant thereof, fragment
thereof, or analog or derivative thereof.
[0511] Generally, the PRODH protein of the present invention may be
derivatized by the attachment of one or more chemical moieties to
the protein moiety. The chemically modified derivatives may be
further formulated for intraarterial, intraperitoneal,
intramuscular subcutaneous, intravenous, oral, nasal, pulmonary,
topical or other routes of administration. Chemical modification of
biologically active component or components may provide additional
advantages under certain circumstances, such as increasing the
stability and circulation time of the component or components and
decreasing immunogenicity. See U.S. Pat. No. 4,179,337, Davis et
al., issued Dec. 18, 1979. For a review, see Abuchowski et al., in
Enzymes as Drugs (J. S. Holcerberg and J. Roberts, eds. pp. 367-383
(1981)). A review article describing protein modification and
fusion proteins is Francis, 1992, Focus on Growth Factors 3:4-10,
Mediscript: Mountview Court, Friem Barnet Lane, London N20, OLD,
UK.
[0512] Chemical Moieties For Derivatization.
[0513] The chemical moieties suitable for denvatization may be
selected from among water soluble polymers. The polymer selected
should be water soluble so that the component to which it is
attached does not precipitate in an aqueous environment, such as a
physiological environment. Preferably, for therapeutic use of the
end-product preparation, the polymer will be pharmaceutically
acceptable. One skilled in the art will be able to select the
desired polymer based on such considerations as whether the
polymer/component conjugate will be used therapeutically, and if
so, the desired dosage, circulation time, resistance to
proteolysis, and other considerations. For the present component or
components, these may be ascertained using the assays provided
herein.
[0514] The water soluble polymer may be selected from the group
consisting of, for example, polyethylene glycol, copolymers of
ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,
polyvinyl alcohol, polyvinyl pyrrolidone, poly-.sup.1, 3-dioxolane,
poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer,
polyaminoacids (either homopolymers or random copolymers), and
dextran or poly(n-vinyl pyrrolidone)polyetlylene glycol,
propropylene glycol homopolymers, prolypropylene oxide/ethylene
oxide co-polymers, polyoxyethylated polyols and polyvinyl alcohol.
Polyethylene glycol propionaldenhyde may have advantages in
manufacturing due to its stability in water.
[0515] The polymer may be of any molecular weight, and may be
branched or unbranched. For polyethylene glycol, the preferred
molecular weight is between about 2 kDa and about 100 kDa (the term
"about" indicating that in preparations of polyethylene glycol,
some molecules will weigh more, some less, than the stated
molecular weight) for ease in handling and manufacturing. Other
sizes may be used, depending on the desired therapeutic profile
(e.g., the duration of sustained release desired, the effects, if
any on biological activity, the ease in handling, the degree or
lack of antigenicity and other known effects of the polyethylene
glycol to a therapeutic protein or analog).
[0516] The number of polymer molecules so attached may vary, and
one skilled in the art will be able to ascertain the effect on
function. One may mono-derivatize, or may provide for a di-, tri-,
tetra- or some combination of derivatization, with the same or
different chemical moieties (e.g., polymers, such as different
weights of polyethylene glycols). The proportion of polymer
molecules to component or components molecules will vary, as will
their concentrations in the reaction mixture. In general, the
optimum ratio (in terms of efficiency of reaction in that there is
no excess unreacted component or components and polymer) will be
determined by factors such as the desired degree of derivatization
(e.g., mono, di-, tri-, etc.), the molecular weight of the polymer
selected, whether the polymer is branched or unbranched, and the
reaction conditions.
[0517] The polyethylene glycol molecules (or other chemical
moieties) should be attached to the component or components with
consideration of effects on functional or antigenic domains of the
protein. There are a number of attachment methods available to
those skilled in the art, e.g., EP 0 401 384 herein incorporated by
reference (coupling PEG to G-CSF), see also Malik et al., 1992,
Exp. Hematol. 20:1028-1035 (reporting pegylation of GM-CSF using
tresyl chloride). For example, polyethylene glycol may be
covalently bound through amino acid residues via a reactive group,
such as, a free amino or carboxyl group. Reactive groups are those
to which an activated polyethylene glycol molecule may be bound.
The amino acid residues having a free amino group include lysine
residues and the--terminal amino acid residues; those having a free
carboxyl group include aspartic acid residues glutamic acid
residues and the C-terminal amino acid residue. Sulfhydryl groups
may also be used as a reactive group for attaching the polyethylene
glycol molecule(s). Preferred for therapeutic purposes is
attachment at an amino group, such as attachment at the N-terminus
or lysine group.
[0518] One may specifically desire N-terminally chemically modified
protein. Using polyethylene glycol as an illustration of the
present compositions, one may select from a variety of polyethylene
glycol molecules (by molecular weight, branching, etc.), the
proportion of polyethylene glycol molecules to protein (or peptide)
molecules in the reaction mix, the type of pegylation reaction to
be performed, and the method of obtaining the selected N-terminally
pegylated protein. The method of obtaining the N-terminally
pegylated preparation (i.e., separating this moiety from other
monopegylated moieties if necessary) may be by purification of the
N-terminally pegylated material from a population of pegylated
protein molecules. Selective N-terminal chemically modification may
be accomplished by reductive alkylation which exploits differential
reactivity of different types of primary amino groups (lysine
versus the N-terminal) available for derivatization in a particular
protein. Under the appropriate reaction conditions, substantially
selective derivatization of the protein at the N-terminus with a
carbonyl group containing polymer is achieved. For example, one may
selectively N-terminally pegylate the protein by performing the
reaction at pH which allows one to take advantage of the pK.sub.1
differences between the e-amino groups of the lysine residues and
that of the a-amino group of the N-terminal residue of the protein.
By such selective derivatization attachment of a water soluble
polymer to a protein is controlled: the conjugation with the
polymer takes place predominantly at the N-terminus of the protein
and no significant modification of other reactive groups, such as
the lysine side chain amino groups, occurs. Using reductive
alkylation, the water soluble polymer may be of the type described
above, and should have a single reactive aldehyde for coupling to
the protein. Polyethylene glycol proprionaldehyde containing a
single reactive aldehyde, may be used.
Pharmaceutical Compositions
[0519] In yet another aspect of the present invention, provided are
pharmaceutical compositions comprising a protein comprising an
amino acid sequence of SEQ ID NO:2, conservative variants thereof,
fragments thereof, or analogs or derivatives thereof, and a
pharmaceutically acceptable carrier thereof. Such pharmaceutical
compositions may be for administration for injection, or for oral,
pulmonary, nasal or other forms of administration. In general,
comprehended by the invention are pharmaceutical compositions
comprising effective amounts of a low molecular weight component or
components, or derivative products, of the invention together with
pharmaceutically acceptable diluents, preservatives, solubilizers,
emulsifiers, adjuvants and/or carriers. Such compositions include
diluents of various buffer content (e.g., Tris-HCl, acetate,
phosphate), pH and ionic strength; additives such as detergents and
solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants
(e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g.,
Thimersol, benzyl alcohol) and bulking substances (e.g., lactose,
mannitol); incorporation of the material into particulate
preparations of polymeric compounds such as polylactic acid,
polyglycolic acid. etc. or into liposomes. Hylauronic acid may also
be used. Such compositions may influence the physical state,
stability, rate of in vivo release, and rate of in vivo clearance
of the present proteins and derivatives. See, e.g., Remington's
Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co.,
Easton, Pa. 18042) pages 1435-1712 which are herein incorporated by
reference. The compositions may be prepared in liquid form, or may
be in dried powder, such as lyophilized form.
Oral Delivery
[0520] Contemplated for use herein are oral solid dosage forms,
which are described generally in Remington's Pharmaceutical
Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa. 18042) at
Chapter 89, Which is herein incorporated by reference. Solid dosage
forms include tablets, capsules, pills, troches or lozenges,
cachets or pellets. Also, liposomal or proteinoid encapsulation may
be used to formulate the present compositions (as, for example,
proteinoid microspheres reported in U.S. Pat. No. 4,925,673).
Liposomal encapsulation may be used and the liposomes may be
derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556).
A description of possible solid dosage forms for the therapeutic is
given by Marshall, K. In: Modern Pharmaceutics Edited by G. S.
Banker and C. T. Rhodes Chapter 10, 1979, herein incorporated by
reference. In general, the formulation will include the component
or components (or chemically modified forms thereof) and inert
ingredients which allow for protection against the stomach
environment, and release of the biologically active material in the
intestine.
[0521] Also specifically contemplated are oral dosage forms of the
above derivatized PRODH, conservative variant thereof, or fragment
thereof. The PRODH, conservative variant thereof, or fragment
thereof may be chemically modified so that oral delivery of the
derivative is efficacious. Generally, the chemical modification
contemplated is the attachment of at least one moiety to the
component molecule itself, where said moiety permits (a) inhibition
of proteolysis; and (b) uptake into the blood stream from the
stomach or intestine. Also desired is the increase in overall
stability of the component or components and increase in
circulation time in the body. Examples of such moieties include:
polyethylene glycol, copolymers of ethylene glycol and propylene
glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol,
polyvinyl pyrrolidone and polyproline. Abuchowski and Davis, 1981,
"Soluble Polymer-Enzyme Adducts" In: Enzymes as Drugs, Hocenberg
and Roberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-383;
Newmark, et al., 1982, J. Appl. Biochem. 4:185-189. Other polymers
that could be used are poly-13-dioxolane and poly-1,3,6-tioxocane.
Preferred for pharmaceutical usage, as indicated above, are
polyethylene glycol moieties.
[0522] For the component (or derivative) the location of release
may be the stomach, the small intestine (the duodenum, the jejunum,
or the ileum), or the large intestine. One skilled in the art has
available formulations which will not dissolve in the stomach, yet
will release the material in the duodenum or elsewhere in the
intestine. Preferably, the release will avoid the deleterious
effects of the stomach environment, either by protection of the
protein (or derivative) or by release of the biologically active
material beyond the stomach environment, such as in the
intestine.
[0523] To ensure fill gastric resistance a coating impermeable to
at least pH 5.0 is essential. Examples of the more common inert
ingredients that are used as enteric coatings are cellulose acetate
trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP),
HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit
L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L,
Eudragit S, and Shellac. These coatings may be used as mixed
films.
[0524] A coating or mixture of coatings can also be used on
tablets, which are not intended for protection against the stomach.
This can include sugar coatings, or coatings which make the tablet
easier to swallow. Capsules may consist of a hard shell (such as
gelatin) for delivery of dry therapeutic i.e. powder; for liquid
forms, a soft gelatin shell may be used. The shell material of
cachets could be thick starch or other edible paper. For pills,
lozenges, molded tablets or tablet triturates, moist massing
techniques can be used.
[0525] The therapeutic can be included in the formulation as fine
multi-particulates in the form of granules or pellets of particle
size about 1 mm. The formulation of the material for capsule
administration could also be as a powder, lightly compressed plugs
or even as tablets. The therapeutic could be prepared by
compression.
[0526] Colorants and flavoring agents may all be included. For
example, the protein (or derivative) may be formulated (such as by
liposome or microsphere encapsulation) and then further contained
within an edible product, such as a refrigerated beverage
containing colorants and flavoring agents.
[0527] One may dilute or increase the volume of the therapeutic
with an inert material. These diluents could include carbohydrates,
especially mannitol, a-lactose, anhydrous lactose, cellulose,
sucrose, modified dextrans and starch. Certain inorganic salts may
be also be used as fillers including calcium triphosphate,
magnesium carbonate and sodium chloride. Some commercially
available diluents are Fast-Flo, Emdex. STA-Rx 1500, Emcompress and
Avicell.
[0528] Disintegrants may be included in the formulation of the
therapeutic into a solid dosage form. Materials used as
disintegrates include but are not limited to starch, including the
commercial disintegrant based on starch, Explotab. Sodium starch
glycolate, Amberlite, sodium carboxymethylcellulose,
ultramylopectin, sodium alginate, gelatin, orange peel acid
carboxymethyl cellulose, natural sponge and bentonite may all be
used. Another form of the disintegrants are the insoluble cationic
exchange resins. Powdered gums may be used as disintegrants and as
binders and these can include powdered gums such as agar, Karaya or
tragacanth. Alginic acid and its sodium salt are also useful as
disintegrants.
[0529] Binders may be used to hold the therapeutic agent together
to form a hard tablet and include materials from natural products
such as acacia, tragacanth, starch and gelatin. Others include
methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl
cellulose (CMC). Polyvinyl pyrrolidone (PVP) and
hydroxypropylmethyl cellulose (HPMC) could both be used in
alcoholic solutions to granulate the therapeutic.
[0530] An anti-frictional agent may be included in the formulation
of the therapeutic to prevent sticking during the formulation
process. Lubricants may be used as a layer between the therapeutic
and the die wall, and these can include but are not limited to;
stearic acid including its magnesium and calcium salts,
polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and
waxes. Soluble lubricants may also be used such as sodium lauryl
sulfate, magnesium lauryl sulfate, polyethylene glycol of various
molecular weights, Carbowax 4000 and 6000.
[0531] Glidants that might improve the flow properties of the drug
during formulation and to aid rearrangement during compression
might be added. The glidants may include starch, talc, pyrogenic
silica and hydrated silicoaluminate.
[0532] To aid dissolution of the therapeutic into the aqueous
environment a surfactant might be added as a wetting agent.
Surfactants may include anionic detergents such as sodium lauryl
sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium
sulfonate. Cationic detergents might be used and could include
benzalkonium chloride or benzethomium chloride. The list of
potential non-ionic detergents that could be included in the
formulation as surfactants are lauromacrogol 400, polyoxyl 40
stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60,
glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty
acid ester, methyl cellulose and carboxymethyl cellulose. These
surfactants could be present in the formulation of the protein or
derivative either alone or as a mixture in different ratios.
[0533] Additives which potentially enhance uptake of the protein
(or derivative) are for instance the fatty acids oleic acid,
linoleic acid and linolenic acid.
[0534] Controlled release oral formulation may be desirable. The
drug could be incorporated into an inert matrix which permits
release by either diffusion or leaching mechanisms, e.g., gums.
[0535] Slowly degenerating matrices may also be incorporated into
the formulation. Some enteric coatings also have a delayed release
effect. Another form of a controlled release of this therapeutic is
by a method based on the Oros therapeutic system (Alza Corp.), i.e.
the drug is enclosed in a semipermeable membrane which allows water
to enter and push drug out through a single small opening due to
osmotic effects.
[0536] Other coatings may be used for the formulation. These
include a variety of sugars which could be applied in a coating
pan. The therapeutic agent could also be given in a film coated
tablet and the materials used in this instance are divided into 2
groups. The first are the nonenteric materials and include methyl
cellulose, ethyl cellulose, Methydroxyethyl cellulose,
methylhydroxyethyl cellulose, hydroxypropyl cellulose,
hydroxypropyl-methyl cellulose, sodium carboxymethyl cellulose,
providone and the polyethylene glycols. The second group consists
of the enteric materials that are commonly esters of phthalic
acid.
[0537] A mix of materials might be used to provide the optimum film
coating. Film coating may be carried out in a pan-coater or in a
fluidized bed or by compression coating.
[0538] Pulmonary Delivery.
[0539] Also contemplated herein is pulmonary delivery of the
present protein (or derivatives thereof). The protein (or
derivative) is delivered to the lungs of a mammal while inhaling
and traverses across the lung epithelial lining to the blood
stream. Other reports of this include Adjei et al., 1990,
Pharmaceutical Research, 7:565-569; Adjei et al., 1990,
International Journal of Pharmaceutics, 63:135-144 (leuprolide
acetate); Braquet et al., 1989, Journal of Cardiovascular
Pharmacology, 13(suppl. 5):143-146 (endothelin-l). Hubbard et al.,
1989, Annals of Internal Medicine, Vol. III, pp. 206-212
(a1-antitryspin); Smith et al., 1989, J. Clin. Invest. 84:1145-1146
(a-1-proteinase); Oswein et al., 1990, "Aerosolization of
Proteins", Proceedings of Symposium on Respiratory Drug Delivery
II, Keystone, Colo., March, (recombinant human growth hormone);
Debs et al., 1988, J. Immunol. 140:3482-3488 (interferon-g and
tumor necrosis factor alpha) and Platz et al. U.S. Pat. No.
5,284,656 (granulocyte colony stimulating factor). A method and
composition for pulmonary delivery of drugs for systemic effect is
described in U.S. Pat. No. 5,451,569, issued Sep. 19, 1995 to Wong
et al.
[0540] Contemplated for use in the practice of this invention are a
wide range of mechanical devices designed for pulmonary delivery of
therapeutic products, including but not limited to nebulizers,
metered dose inhalers, and powder inhalers, all of which are
familiar to those skilled in the art.
[0541] Some specific examples of commercially available devices
suitable for the practice of this invention are the Ultravent
nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the
Acorn II nebulizer, manufactured by Marquest Medical Products,
Englewood, Colo.; the Ventolin metered dose inhaler, manufactured
by Glaxo Inc., Research Triangle Park, North Carolina; and the
Spinhaler powder inhaler, manufactured by Fisons Corp. Bedford,
Mass.
[0542] All such devices require the use of formulations suitable
for the dispensing of protein (or derivative). Typically, each
formulation is specific to the type of device employed and may
involve the use of an appropriate propellant material, in addition
to the usual diluents, adjuvants and/or carriers useful in therapy.
Also, the use of liposomes, microcapsules or microspheres,
inclusion complexes, or other types of carriers is contemplated.
Chemically modified protein may also be prepared in different
formulations depending on the type of chemical modification or the
type of device employed.
[0543] Formulations suitable for use with a nebulizer, either jet
or ultrasonic, will typically comprise protein (or derivative)
dissolved in water at a concentration of about 0.1 to 25 mg of
biologically active protein per mL of solution. The formulation may
also include a buffer and a simple sugar (e.g., for protein
stabilization and regulation of osmotic pressure). The nebulizer
formulation may also contain a surfactant, to reduce or prevent
surface induced aggregation of the protein caused by atomization of
the solution in forming the aerosol.
[0544] Formulations for use with a metered-dose inhaler device will
generally comprise a finely divided powder containing the protein
(or derivative) suspended in a propellant with the aid of a
surfactant. The propellant may be any conventional material
employed for this purpose, such as a chlorofluorocarbon, a
hydrochlorofluorocarbon, a hydrotluorocarbon, or a hydrocarbon,
including trichlorofluoromethane, dichlorodifluoromethane,
dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or
combinations thereof. Suitable surfactants include sorbitan
trioleate and soya lecithin. Oleic acid may also be useful as a
surfactant.
[0545] Formulations for dispensing from a powder inhaler device
will comprise a finely divided dry powder containing protein (or
derivative) and may also include a bulking agent, such as lactose,
sorbitol, sucrose, or mannitol in amounts which facilitate
dispersal of the powder from the device, e.g., 50 to 90% by weight
of the formulation. The protein (or derivative) should most
advantageously be prepared in particulate form with an average
particle size of less than 10 nm (or microns), most preferably 0.5
to 5 mm, for most effective delivery to the distal lung.
[0546] Nasal Delivery.
[0547] Nasal delivery of the protein (or derivative) is also
contemplated. Nasal delivery allows the passage of the protein to
the blood stream directly after administering the therapeutic
product to the nose, without the necessity for deposition of the
product in the lung.
[0548] Formulations for nasal delivery include those with dextran
or cyclodextran.
Methods of Treatment, Methods of Preparing a Medicament
[0549] In another embodiment, the present invention extends to a
method of treating schizophrenia or a disease or disorder related
thereto, comprising administering to a mammal a therapeutically
effective amount of a material selected from the group consisting
of a PRODH comprising an amino acid sequence of SEQ ID NO:2, a
conservative variant thereof, a fragment thereof, or an analog or
derivative thereof. The phrase "therapeutically effective amount"
is used herein to mean an amount sufficient to treat, and
preferably decrease by at least about 30 percent, more preferably
by at least 50 percent, most preferably by at least 90 percent, a
symptom or symptoms associated with schizophrenia or a disease or
disorder related thereto in a subject. In a particular embodiment,
the PRODH, conservative variants thereof, fragments thereof, or
analogs or derivatives thereof are delivered parenterally as
described above.
[0550] In yet another aspect of the present invention, methods of
treatment and manufacture of a medicament are provided. Conditions
alleviated or modulated by the administration of the present
derivatives are those indicated above.
[0551] Dosages.
[0552] For all of the above molecules, as further studies are
conducted, information will emerge regarding appropriate dosage
levels for treatment of various conditions in various patients, and
the ordinary skilled worker, considering the therapeutic context,
age and general health of the recipient, will be able to ascertain
proper dosing.
Methods of Assaying Drugs or Agents Using Mice and the PPI Test
[0553] Disclosed infra is a new mouse strain useful in assaying
drugs or agents for the ability to treat schizophrenia, and the
teachings to produce such a mouse. Furthermore, the PPI test which
is used to examine the mice is discussed in detail below.
[0554] Hence, the present extends to a method of identifying drugs
or agents useful in treating schizophrenia or a disease or disorder
related thereto comprising the steps of:
[0555] a) performing an first pre-pulse inhibition test (PPI) test
on a mouse having within its genome two copies of an isolated
variant allele of a Prodh gene comprising a DNA sequence of SEQ ID
NO:7, wherein both copies are capable of expressing a mutant Prodh
comprising an amino acid sequence of SEQ ID NO:8, to obtain a first
percentage of inhibition of startle response;
[0556] b) administering the potential drug or agent to the
mouse;
[0557] c) performing a second PPI test on the mouse to obtain a
second percentage of inhibition of startle response; and
[0558] d) comparing the first percentage to the inhibition of
startle response with the second percentage of startle
response,
[0559] wherein an increase in percentage of inhibition in the
second percentage of inhibition relative to the first percentage of
inhibition is indicative of the ability of the drug or agent to
treat schizophrenia or a disease or disorder related thereto. Thus,
if the percentage of inhibition of startle response in the mouse
having within its two active copies of an isolated variant allele
of a Prodh gene comp mouse after administration of the drug or
agent is greater than the percentage of inhibition of startle
response in the Pro/Re mouse prior to inhibition, then the drug or
agent has the ability to treat schizophrenia or a disease or
disorder related thereto, such as obsessive compulsive disorder
(OCD), bipolar disorder (BP) or major depressive disorder.
[0560] Also, the present invention extends to a method for
identifying a drug or agent for treating schizophrenia or a disease
or disorder related thereto. An example of such a method comprises
the steps of:
[0561] performing an first pre-pulse inhibition test (PPI) test on
an F3 generation mouse from a cross Pro/Re X C57B1/6J wild-type,
wherein the F3 generation mouse has two copies within its genome of
an isolated variant allele of a Prodh gene comprising a DNA
sequence of SEQ ID NO:7 which are capable of expressing a mutant
Prodh comprising an amino acid sequence of SEQ ID NO:8, to obtain a
first percentage of inhibition of startle response;
[0562] administering the potential drug or agent to the F3
generation mouse from a cross of Pro/Re X C57B1/6J wild-type;
[0563] performing a second PPI test on the F3 generation mouse from
a cross of Pro/Re X C57B1/6J wild-type to obtain a second
percentage of inhibition of startle response; and comparing the
first percentage to the inhibition of startle response with the
second percentage of startle response,
[0564] wherein an increase in percentage of inhibition in the
second percentage of inhibition relative to the first percentage of
inhibition is indicative of the ability of the drug or agent to
treat schizophrenia or a disease or disorder related thereto.
[0565] What's more, the present invention extends to a method for
identifying a drug or agent for use in treating schizophrenia or a
disease or disorder related thereto, comprising the steps of:
[0566] administering the drug or agent to an F3 generation mouse
from a cross of Pro/Re X C57B1/6J wild-type, wherein the F3
generation mouse has two copies within its genome of an isolated
variant allele of a Prodh gene comprising a DNA sequence of SEQ ID
NO:7 which are capable of expressing a mutant Prodh comprising an
amino acid sequence of SEQ ID NO:8;
[0567] performing a PPI test on the F3 generation mouse from a
cross of Pro/Re X C57B1/6J wild-type to obtain a percentage of
inhibition of the startle response in the F3 generation mouse from
a cross of Pro/Re X C57B1/6J wild-type which was administered the
drug or agent; and
[0568] comparing the percentage of inhibition of the startle
response in the F3 generation mouse from a cross of Pro/Re X
C57B1/6J wild-type with the percentage of inhibition of the startle
response in an unmedicated F3 generation mouse from a cross of
Pro/Re X C57B1/6J wild-type, wherein the F3 generation mouse has
two copies within its genome of an isolated variant allele of a
Prodh gene comprising a DNA sequence of SEQ ID NO:7 which are
capable of expressing a mutant Prodh comprising an amino acid
sequence of SEQ ID NO:8.
[0569] An increase in percentage of inhibition in the percentage of
inhibition in the medicated mouse relative to the percentage of
inhibition in the unmedicated mouse is indicative of the ability of
the drug or agent to treat schizophrenia or a disease or disorder
related thereto.
[0570] Furthermore, the present invention extends to a method for
identifying a drug or agent for use in treating schizophrenia or a
disease or disorder related thereto, comprising the steps of:
[0571] administering the drug or agent to a mouse having within its
genome two copies of an isolated variant allele of a Prodh gene
comprising a DNA sequence of SEQ ID NO:7, wherein both copies are
capable of expressing a mutant Prodh comprising an amino acid
sequence of SEQ ID NO:8;
[0572] performing a PPI test on the mouse to obtain a percentage of
inhibition of the startle response in the mouse; and
[0573] comparing the percentage of inhibition of the startle
response in the mouse with the percentage of inhibition of the
startle response in an unmedicated mouse having within its genome
two copies of an isolated variant allele of a Prodh gene comprising
a DNA sequence of SEQ ID NO:7, wherein both copies are capable of
expressing a mutant Prodh comprising an amino acid sequence of SEQ
ID NO:8.
[0574] An increase in percentage of inhibition in the percentage of
inhibition in the medicated mouse relative to the percentage of
inhibition in the unmedicated mouse is indicative of the ability of
the drug or agent to treat schizophrenia or a disease or disorder
related thereto.
[0575] In addition, the present invention extends to a method for
identifying a drug or agent for use in treating schizophrenia or a
disease or disorder related thereto, comprising the steps of:
[0576] administering the drug or agent to a mouse having within its
genome two copies of an isolated variant allele of a Prodh gene
comprising a DNA sequence of SEQ ID NO:7, wherein both copies are
capable of expressing a mutant Prodh comprising an amino acid
sequence of SEQ ID NO:8;
[0577] performing a PPI test on the mouse to obtain a percentage of
inhibition of the startle response in the mouse; and
[0578] comparing the percentage of inhibition of the startle
response in the mouse with the percentage of inhibition of the
startle response in an unmedicated mouse having within its genome
two copies of an isolated Prodh gene comprising a DNA sequence of
SEQ ID NO:3, wherein both copies are capable of expressing a Prodh
comprising an amino acid sequence of SEQ ID NO:4.
[0579] If the percentage of inhibition of the startle response in
the medicated mouse is statistically equivalent to the percentage
of inhibition in the mouse capable of expressing Prodh comprising a
DNA sequence of SEQ ID NO:4, then the drug or agent has the ability
to treat schizophrenia or a disease or disorder related
thereto.
[0580] In another embodiment, the present invention extends to an a
method for identifying a drug or agent for use in treating
schizophrenia or a disease or disorder related thereto, comprising
the steps of:
[0581] administering the drug or agent to an F3 generation mouse
from a cross of Pro/Re X C57B1/6J wild-type, wherein the F3
generation mouse has two copies within its genome of an isolated
variant allele of a Prodh gene comprising a DNA sequence of SEQ ID
NO:7 which are capable of expressing a mutant Prodh comprising an
amino acid sequence of SEQ ID NO:8;
[0582] performing a PPI test on the F3 generation mouse from a
cross of Pro/Re X C57B1/6J wild-type administered the drug or agent
to obtain a percentage of inhibition of the startle response in the
mouse; and
[0583] comparing the percentage of inhibition of the startle
response in F3 generation mouse from a cross of Pro/Re X C57B1/6J
wild-type administered the drug with the percentage of inhibition
of the startle response in an F3 generation mouse from a cross of
Pro/Re X C57B1/6J wild-type, wherein the F3 generation mouse has
two copies within its genome of an isolated Prodh gene comprising a
DNA sequence of SEQ ID NO:3 which are capable of expressing a Prodh
comprising an amino acid sequence of SEQ ID NO:4
[0584] If the percentage of inhibition of the startle response in
the medicated mouse is statistically equivalent to the percentage
of inhibition in the mouse capable of expressing Prodh comprising a
DNA sequence of SEQ ID NO:4, then the drug or agent has the ability
to treat schizophrenia or a disease or disorder related
thereto.
[0585] The PPI test involves the evaluation of sensimotor gating.
In particular, this test permits the evaluation of sensormotor
gating from the degree of inhibition of an acoustic startle
response by a prepulse preceding by 100 msec an abrupt startling
stimulus (pre-pulse inhibition). PPI was recorded using a
combination of two startle levels (100 and 115 dB) and two prepulse
levels (82 and 90 dB) and is expressed as:
100-[(response to startle stimulus following pre-pulse/response to
startle stimulus alone).times.100].
[0586] Testing was conducted in an SR lab system (San Diego
Instruments) Each of two accoustically insulated startle chambers
contained a transparent acrylic cylinder (4 cm in diameter) mounted
on a frame to which a motion sensor was attached for the detection
and transduction of movement, and a sound generation system for the
delivery of background white noise and acoustic stimuli.
Immediately after placement in the chamber, the animal was given a
5 min. Acclimation period during which background noise (67 dB) was
continually present, and then received 6 sets of the following 7
trial types counterbalanced to control for order: Trial 1: 40 ms,
100 dB noise burst alone; trial 2: 40 ms, 115 dB noise burst alone;
trial 3-6: 100 dB or 115 dB startle stimuli preceded 100 ms by a 20
ms, 82 dB or 90 dB noise burst (prepulse); trial 7:
no-stimulus/background noise alone (67 dB). Intertrial interval was
variable (10-20 sec with an average of 15 sec). At the beginning of
the block of 42 trials, the animal received the following 3 trials:
1 no-stimulus/background noise along (67 dB) trial, 1 startle
stimulus alone trial for both 100 dB and 115 dB. At the end of the
block of 42 trials the animal received the same 3 trials again in
reverse order.
[0587] The background noise level was 67 dB during the entire
testing session. Data was analyzed using ANOVA with repeated
measures.
[0588] The present invention may be better understood by reference
to the following non-limiting Example, which is provided as
exemplary of the invention. The following Example is presented in
order to more fully illustrate the preferred embodiments of the
invention. It should in no way be construed, however, as limiting
the broad scope of the invention.
EXAMPLE
[0589] Evidence for an association between schizophrenia
susceptibility and hemizygous deletions in chromosome 22q11 has
previously been determined [Karayiorgou et al., Proc. Natl. Acad.
Sci. U.S.A. 92, 7612 (1995)]. More specifically, three hemizygous
cryptic deletions at 22q11 in a sample of 300 unrelated
schizophrenic patients have previously been reported and
characterized. [M. Karayiorgou et al., Proc. Natl. Acad. Sci.
U.S.A. 92, 7612 (1995); M. Karayiorgou et al., Amer. J. Med. Genet.
74, 677 (1997)] The frequency of this microdeletion in the general
population is estimated to be .sup..about.0.02% (although the
latter is most likely an underestimate) and no deletions were found
in a sample of 200 healthy controls. [M. Karayiorgou et al., Proc.
Natl. Acad. Sci. U.S.A. 92, 7612 (1995); M. Karayiorgou et al.,
Amer. J. Med. Genet. 74, 677 (1997)] The identified locus
(.sup..about.1.5 Mb in size) is located in the proximal part of a
region at chromosome 12q11, and has been implicated independently
in schizophrenia susceptibility through linkage studies.
[Karayiorgou, M. and Gogos, J., Neuron 19, 967 (1997)] This locus
overlaps with a critical region involved in the etiology of
Velocardio-facial (VCFS)/DiGeorge (DGS) syndromes [Driscoll et al.,
J. Med. Genet. 30, 813 (1993)] It has been shown that
.sup..about.29% VCFS children with 22q11 deletions develop
schizophrenia or schizoaffective disorder in adolescence and
adulthood [Pulver, A. E., et al., J. Nerv. Ment. Dis. 182, 476
(1994)] an estimate confirmed by a more recent independent study.
22q11 deletions have been identified among schizophrenia patients
of diverse ethnic origins (Chinese, Israeli, British, Danish) and
one study implicated the 22q11 region in early-onset schizophrenia
Yan et al., Am. J. Med. Genet. 81, 41 (1998)1. In addition, the
increased rates of comorbid obsessive compulsive disorder (OCD) or
symptoms (OCS) among schizophrenic patients with the 22q11
microdeletion locus [Karayiorgou M., Gogos, J A, et al., Genotype
and Phenotype Analysis of the 22q11 Schizophrenia Susceptibility
Locus. Cold Spring Harbor Symposia on Quantitative biology, Vol.
LXI, pp. 835-843, Cold Spring Harbor Press, Cold Spring Harbor,
N.Y. (1996); Karayiorgou, M. et al., Proc. Natl. Acad. Sci. 94,
4572-4575 (1997)] and similarly increased rates of anxiety, OCS and
OCD in children and adults with the 22q11 microdeletion in the
absence of schizophrenia indicates that the 22q11 genomic region
may harbor one or more genes predisposing to schizophrenia or a
disease or disorder related thereto.
[0590] Furthermore, an association in a recessive manner between a
low activity allele of the Catechol-O-methyltransferase (COMT)
gene, located at the proximal part of the 22q11 deleted region, and
a susceptibility to OCD, particularly in males, has been reported.
[Karayiorgou et al. 1997, supra.] The observation that
.sup..about.20% of schizophrenia patients report obsessions and
compulsions, features that are found in only 1-2% of the general
population [Eisen, J. L & Rasmussen, S. A., Obsessive
Compulsive Disorder with Psychotic Features. J. Clin. Psychiatry
54:373-379 (1993); Berman, I. Kalinowski, A., Berman, S. M.,
Lengua, J., and Green, A. I., Obsessive and Compulsive Disorders in
Chronic Schizophrenia. Compr. Psychiatry 36:6-10 (1995)1 indicates
that schizophrenia and OCD may share some pathophysiological and
genetic components. For example, one common central processing
mechanism that seems to be affected in patients with schizophrenia
and OCD is sensorimotor gating. Patients with schizophrenia and OCD
demonstrate poor sensorimotor gating of the startle response as
measured by impaired prepulse inhibition of an acoustic response
and this may lead to sensory overload, distractibility and
cognitive fragmentation.
[0591] Reported herein is the isolation and characterization of the
human homolog (FIG. 1A) of the D. melanogaster slgA proline
dehydrogenase gene (PRODH), which is responsible for the behavioral
phenotype of the D. melanogaster sluggish-A mutant [Hayward, D. C.,
et al., Proc. Natl. Acad. Sci. USA 90, 2979 (1993)]. The gene was
localized at the most centromeric part of the 22q11 deletions and
was shown to be expressed in several tissues, including brain.
Mapping of PRODH indicates this gene may contribute to the
psychiatric phenotype associated with the 22q11 deletions, because
proline has long been suspected to serve as a modulator of a
synaptic transmission in the mammalian brain and in addition,
proline dehydrogenase is involved in the biosynthesis and release
of the neurotransmitter glutamate.
[0592] Elevated levels of proline have been reported in a
DiGeorge/VCFS patient [Jacken, J., Goemans, N. Frynes, J. -P,
Francois, I., de Zegher, F., J. Inherit. Metab. Dis. 19. 275
(1996)], wherein DiGeorge/VCFS is a contiguous gene microdeletion
syndrome involving chromosome 22q11. The de novo origin of the
deletion and the fact that both the parents and the sister of the
affected proband had normal proline levels, suggested that deletion
of a gene located within the 22q11 region is responsible for the
observed hyperprolinaemia. Proline dehydrogenase is the first
enzyme of proline catabolism that converts proline to
.DELTA.1-pyrroline-5-carboxyl- ate [Wang, S. S. and Brandriss, M.
C., Mol. Cell Biol. 6, 2638 (1986); Wang, S. S. and Brandriss, M.
C., Mol. Cell Biol. 7, 4431 (1987)] and dysfunction of this enzyme
is expected to result in abnormal proline metabolism. The
Drosophila melanogaster sluggish-A gene (slgA) was previously shown
to encode a proline dehydrogenase; activity of this enzyme is
abnormally low in the slgA mutant that presents behavior
abnormalities, and is restored to wild-type levels in transgenic
flies carrying the wildtype slgA gene [Hayward, D. C., et al.,
Proc. Natl. Acad. Sci. USA 90, 2979 (1993)]. Database searches
using the sequence of the D. melanogaster slgA gene identified
several human Expressed Sequence Tag (EST) cDNA clones, encoding a
protein sequence with a strong match to the D. melanogaster slgA
coding region. In particular, an insert from EST ym93b08.r1 from a
human brain library (GenBank R88591) was used to screen a human
cerebellar as well as a kidney cDNA library using a BLAST search
provided by the National Institutes of Health at
http://www.ncbi.nlm.nih.- gov/cgi-bin/BLAST on the World Wide Web.
A score having an E value of 4e.sup.-06 is considered a "strong
match". A cDNA fragment was used as a hybridization probe against a
panel of monochromatic hybrids as well as an array of nine phage
artificial chromosomes (PACs) previously mapped and ordered within
the 22q11 region [Carlson, C. et al., Am. J. Hum. Genet. 61, 620
(1997)]. Only chromosome 22 (not shown) and PAC-P457M14 provided a
positive signal (FIG. 1B). This PAC is included in the smallest
22q11 deletion identified to date in patients with schizophrenia
[M. Karayiorgou et al., Proc. Natl. Acad. Sci. U.S.A. 92, 7612
(1995); M. Karayiorgou et al., Amer. J. Med. Genet. 74, 677 (1997)]
suggesting that the human homologue of slgA maps within the deleted
region. Mapping was confirmed by PCR analysis on a previously
described [M. Karayiorgou et al., Proc. Natl. Acad. Sci. U.S.A. 92,
7612 (1995); M. Karayiorgou et al., Amer. J. Med. Genet. 74, 677
(1997)1 somatic cell hybrid line carrying a copy of the deleted
chromosome 22 (not shown). Library screening, Northern blotting and
hybridization, reverse transcription PCR and 5' Rapid Amplification
of cDNA ends (5' RACE) were performed according to standard
protocols [J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular
Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 2nd edition, 1989)]. The sequence of both
human and mouse clones was obtained by primer walking from both
strands. Sequence alignment and estimations of sequence similarity
were performed using the program DNASTAR. 22q11 PAC clones were
provided by P. deJong, Roswell Polytechnical College and Institute,
Buffalo, N.Y. Monochromatic hybrids were purchased from BIOS
Laboratories (New Haven, Conn.).
[0593] FIG. 1A shows an alignment of the predicted human PRODH with
the D. melanogaster SLGA and yeast PUT1 proline dehydrogenase
proteins, as well as with sequence from the mouse homologue (see
below). The human and D. melanogavter proteins show 40.4% overall
similarity as defined in the "DNASTAR LASERGENE" software program
(DNASTAR, Inc. 1228 South Park Street, Madison, Wis. 53715),
although the region of highest similarity among all homologues is
localized toward the C-terminus where the human and D. melanogaster
proteins are 60.4% similar. Northern blot analysis and
autoradiography revealed a 2.4-2.5 kb band in several human tissues
(such as heart, lung, liver, skeletal muscle, kidney, pancreas),
including brain (not shown) and higher resolution analysis of the
brain expression pattern revealed widespread distribution of the
mRNA in several regions (FIG. 1C).
[0594] To determine whether the PRODH gene contributes to
psychiatric phenotypes associated with the 22q11 deletions, two
complementary studies were performed and reported herein. The first
study involves isolation of the mouse Prodh gene, and
identification of a mutation of this gene in the Pro/Re
hypeprolinemic mouse strain. Furthermore, analysis of the startle
response attenuation by prepulse inhibition (PPI) in these mice was
used as a measure of sensorimotor gating, a central processing
mechanism that is affected in patients with schizophrenia and OCD.
The second study involved identification of PRODH gene variants
that can be used to examine their inheritance in nuclear families
with schizophrenia and obsessive compulsive disorder. These studies
are described in more detail below.
[0595] In the first study reported herein, human PRODH cDNA was
used as a probe to isolate and sequence mouse Prodh cDNA from a
cerebellar cDNA library. The mouse and human proteins show 86%
similarity (FIG. 1A). Northern blot analysis and autoradiography
revealed a 2.4-2.5 kb band in several mouse tissues (such as heart,
lung, liver, skeletal muscle, kidney, pancreas) including brain
(FIG. 2A). The PRO/Re mouse [R. L. Blake and E. S. Russell, Science
176, 809 (1972)], wherein the PRO/Re strain is a highly inbred
strain developed by over 25 generation sibling inbreeding starting
from an original cross between 129/ReJ and C57BL/6J strains,
carries a presumably homozygous defect in proline dehydrogenase
activity. In order to understand the contribution of the PRODH gene
in the 22q11 associated psychiatric phenotype, the mouse Prodh gene
from the PRO/Re mice was isolated, and a G to T substitution was
identified 135 nucleotides 5' of the native termination codon has
been identified that results in a premature translational
termination (FIG. 1A, FIG. 2B). This substitution eliminates an
MnlI site, and this information was used for genotyping the F3
generation from a cross between the original Pro/Re and C57/B6 wild
type strain (F2 heterozygotes were intercrossed and offspring were
genotyped by PCR]. Analysis of plasma amino acid levels
demonstrated that presence of the identified mutation correlates
with increased levels of proline in F3 mice, and, thus most likely
accounts for the hyperprolinemia in the Pro/Re Strain.
Specifically, F3 homozygous for the mutation present an average
seven fold increase of the blood proline levels. In contrast,
comparisons of glutamate levels did not reveal any significant
changes (FIG. 2C).
[0596] Adult Prodh-deficient homozygous mice (TAG/TAG) were normal
in appearance and development and had normal viability. However,
2-3 month old male Prodh-deficient homozygous mice weighed
.sup..about.10% less than wild type littermates (GAG/GAG)
[F(1.33)=12.625, p=0. 0012)].
[0597] Brain morphology appeared identical in homozygous mice and
their wild type littermates by gross evaluation. Upon histological
examination of sections, cell groups in the forebrain and
diencephalon appeared to be well formed with no obvious
neuroanatomical alterations. Abnormal proline metabolism may alter
biosynthesis and release of glutamate and y-aminobutyric acid
(GABA) [Johnson, J. L. and Roberts, E., Brain Res. 323, 247 (1984);
Yoneda, Y. and Roberts, E., Brain Res. 239, 479 1982); Yoneda, Y.,
Roberts, E., and Dietz, G. W., J. Neurochem. 38, 1686 (1982)].
Because L-glutamate is the principal neuromuscular transmitter in
D. melanogaster, it has been hypothesized that a deficiency in
proline dehydrogenase may alter L-glutamate metabolism in specific
nerve cell populations in D. melanogaster, thus being responsible
for the sluggish phenotype reported by Hayward et al., (1986). GABA
is thought to be the major inhibitory neurotransmitter of various
inhibitory interneurons. Glutamate, in addition to being a major
excitatory amino acid transmitter, may also serve as a precursor of
GABA and aspartate, another excitatory transmitter [Johnson, et al
(1984). Therefore, basal levels of glutamate, GABA and aspartate
were compared in extracts from frontal cortex, hypothalamus,
amygdala, and hippocampus of homozygous mutant and wild type
animals (FIG. 2D) [Luine, V. N., Grattan, D. R., and Selmanoff, M.,
Brain Res. 747, 165 (1997); Grattan, D. and Selmanoff, M., J.
Neurochem. 60, 2254 (1993)]. The frontal cortex section taken was
between approximately +3.08 and +1.70 mm (according to the atlas of
K. B. J. Franklin and G. Paxinos, The Mouse Brain in Stereotaxic
Coordinates, Academic Press, New York, 1997) and two dorsomedial
punches were taken. It contained the medial and ventral orbital
cortex and the pre- and infra-limbic cortex. One punch of the
dorsal hippocampus was taken between approximately -2.0 and -3.0
mm. The hypothalamus was sampled with bilateral punches between
approximately 0.34 and 2.18 mm and consisted of the dorso and
ventromedial nuclei, anterior hypothalamic nucleus, and arcuate
nucleus. Aminoacids glutamate, aspartate, glycine, serine and
.gamma.-aminobutyric acid (GABA)- in the brain samples were
measured using HPLC with electrochemical detection following
precolumn derivatization of the sample with 0-phthalaldehyde and
beta-mercaptoethanol. Briefly, punched tissues were expelled into
sodium acetate buffer, pH 5.0, containing approximately 25 ng of
homoserine as an internal standard (150 .mu.l for cortex and 200
.mu.l for hippocampus). The samples were centrifuged and 15 .mu.l
of supernatant, following automated precolumn derivatization, was
injected using a refrigerated, Waters 717 Autosampler onto a
Brownlee Velosep RP-18 3 micron column. Amino acids were detected
with an ESA model 5200 Coulochem II detector with a model 5011
analytical cell with electrodes set at +0.1 mV to oxidize and
remove derivatization contaminants and at +0.45 mV to oxidize and
detect derivatized amino acids. Details of the mobile phase and
derivatization reagent can be found in Grattan and Selmanoff, 1993.
Picograms of amino acids were calculated with the Waters Millenium
computer system by comparing the ratio of the peak heights of the
transmitters and the internal standard in sample chromatograms with
ratio of peak heights in chromatograms from external amino acid
standards. Proteins were determined in the sample pellets and
concentrations of amino acids are expressed as ng/ug of protein.
Measurements were made in a single cohort of mice consisting of
male and female homozygous (n=15) and wild type (n=1).
[0598] An 8% decrease in the levels of glutamate was observed in
the frontal cortex of the Prodh-deficient mice but this did not
reach statistical significance (90.7.+-.5.6 versus 98.8.+-.10.1,
p=0.58). However, a significant .sup.-25% decrease of GABA levels
(5.7.+-.0.3 versus 7.7.+-.0.6, p=0.03) and a .sup.-18% decrease of
the aspartate levels (9.5.+-.0.5 versus 11.5.+-.0.4, p=0.01) was
observed in the same brain region. Similarly, significant decreases
in the levels of glutamate (.sup.-13%, 36.9.+-.1.0 verses
42.3.+-.1.4, p=0.0062), GABA (.sup..about.10%, 9.3.+-.0.1 verses
10.4.+-.0.4, p=0.01) and aspartate (.sup.-10%, 8.0.+-.0.2 verses
8.9.+-.0.3, p=0.03) were observed in the hypothalamus of homozygous
mutants. By contrast, no significant differences were observed
among animals of either genotype in the levels of these three
neurotransmitters in the amygdala and hippocampus. In addition, no
significant differences were observed in the levels of glycine
(another inhibitory neurotransmitter), in all brain regions
examined.
[0599] Homozygous mutant mice and wild type littermates, 2-3 months
old at the onset of testing were analyzed in behavioral assays of
sensory reception/processing, locomotion and anxiety. Forty two
homozygous and 26 wild type littermate mice of both sexes, 2-3
months old at the onset of testing were used for the behavioral
analysis. Animals were housed in groups of 2-4 and were maintained
on a reverse 12:12 h light-dark cycle with lights off at 0700 hrs.
All testing occurred between 0900 and 1800 hrs. Prior to all
testing, animals were handled and weighed. The behavioral tests
were performed in the following order: locomotor activity,
light/dark transition, PPI and habituation of the startle response.
At the conclusion of all behavioral assays the genotypes of all
mice were reconfirmed by PCR analysis. One central processing
mechanism that is affected in patients with psychiatric disorders
(primarily schizophrenia, and probably OCD) is sensorimotor gating,
a neural filtering process that allows attention to be focused on a
given stimulus. This impaired attentional filtering is thought to
result in sensory overload, distractibility, cognitive
fragmentation and possibly psychotic symptoms [D. Braff et al.,
Psychophysiology 15, 339 (1978); C. Grillon, R. Ameli, D. S.
Charney, Biol. Psychiatry 32, 939 (1992); N. R. Swerdlow, C. H.
Benbow, S. Zisook, Biol. Psychiatry 33, 298 (1993); W. Perry and D.
L. Braff, Am. J. Psychiatry 151, 363-367 (1994)1. Sensorimotor
gating can be evaluated from the degree of inhibition of an
acoustic startle response by a weak prepulse preceding by 30-500
msec an abrupt startling stimulus (FIG. 3A, pre-pulse inhibition,
(PPI) [R. Paylor and J. N. Crawley, Psychopharmacology 132, 169
(1997); N. R. Swerdlow, D. L. Braff, N. Taaid, M. A. Geyer, Arch.
Gen. Psychiatry 51, 139 (1994); S. F. Logue, E. H. Owen, D. L.
Rasmussen, J. M. Wehner, Neuroscience 80, 1075 (1997); A. E.
Bullock, B. S. Slobe, V. Vasquez, A. C. Collins, Behav Neurosci.
111, 1353 (1997)]. PPI is one of a few neuropsychological measures
in which humans and rodents can be evaluated more or less in a
similar fashion. In rodents, the neural circuit underlying the
startle reflex is thought to involve four to five synapses [M.
Davis, D. S. Gendelman, M. D. Tischler, P. M. Gendelman, J.
Neurosci. 2, 791 (1982)] and a number of centers and
neurotransmitters may exert prepulse influences on its function [M.
A. Geyer and D. L. Braff, Schizophr. Bull. 13, 643 (1987)]. In
addition, it has been suggested that GABA-mediated presynaptic
control of excitatory neurotransmitter release may be involved in
the control of gating of sensory responses [Duter and R. A. Nicoll,
Neuron 1, 585 (1988)] and GABAergic circuitry may partly mediate
the decrease in sensorimotor gating induced by dopamine
overactivity [N. R. Swerdlow, D. L. Braff, M. A. Geyer, Brain Res.
532, 146 (1990); M. H. Kodsi and N. R. Swerdlow, Brain Res. Bull.
43, 219 (1997)] and activation of nicotinic receptors [N. R.
Swerdlow, D. L. Braff, M. A. Geyer, Brain Res. 532, 146 (1990); M.
H. Kodsi and N. R. Swerdlow, Brain Res. Bull. 43, 219 (1997)].
[0600] Another measure of sensorimotor gating is based on
electoencephalographic techniques designed to study response to
paired auditory stimuli. In this case, suppression of the P50 wave
in humans and the N40 wave in rodents by a second stimulus, when
two auditory stimuli are presented in sequence, is used as a
measure of normal gating [R. Freedman et al., in Schizophrenia:
Origins, processes, treatment, and outcome, R. L. Cromwell and C.
R. Snyder, Eds., (Oxford Univ. Press, New York, 1993), pp. 98-108;
K. E. Stevens et al., Neuropsychopharmacology 15, 152 (1996)].
Patients with schizophrenia as well as some of their
non-symptomatic family members show decreased attenuation of the
second P50 wave. Recently a genetic linkage has been described
between a DNA marker on chromosome 15 and decreased P50 attenuation
in some families with schizophrenia [R. Freedman et al., Proc.
Natl. Acad. Sci. U.S.A. 94, 587 (1997)].
Methods and Materials
Library Screening
[0601] Library screening, Northern blotting and hybridization,
reverse transcription PCR and 5' Rapid Amplification of cDNA ends
(5' RACE) were performed according to standard protocols [J.
Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning: A
Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 2nd edition, 1989)]. The sequence of both human and
mouse clones was obtained by primer walking from both strands.
Sequence alignment and estimations of sequence similarity were
performed using the program DNASTAR. 22q11 PAC clones were provided
by P. deJong, Roswell Polytechnical College and Institute, Buffalo,
N.Y. Monochromatic hybrids were purchased from BIOS Laboratories
(New Haven, Conn.).
Genotyping Mutants and Wild Type Littermates
[0602] Pro/Re and C57/B6 mice were purchased from Jackson
Laboratories. For PCR genotyping for the identified mutation, the
following primers were used:
[0603] F: 5'GACCAAATCAGCTTCCCACT (SEQ ID NO:5);
[0604] R: 5'CCCTTCATGATGCTGCTGTT (SEQ ID NO:6).
[0605] Target sequences were amplified in a 50 Al reaction mixture
containing 100 ng of genomic DNA in 50 mM KCl, 10 mM Tris-HCl (pH
8.3), 1.5 mM MgCl.sub.2, 0.1% gelatin, 50 mm of each primer, 200 mM
of each dNTP (dATP, dCTP, dGTP, dTTP), and 1.5 u Taq polymerase.
PCR was performed in a programmable PCR apparatus (MJ Research,
Inc.). Amplification was as follows: 80.degree. C..times.8 min
(1.times.), 92.degree. C..times.1 min/60.degree. C. .times.40
sec/72.degree. C..times.1 min (30.times.), 72.degree. C. .times.7
min (1.times.), 4.degree. C..times.5 min. 45 .mu.l of the amplified
product was digested with 20-40 .mu. of MnlI in a 60 .mu.l reaction
volume, according to the manufacturer's specifications. The
digested product was electrophoresed in a 4% 1.times. TBE NuSieve
agarose gel.
[0606] Because of the development of a blood-brain barrier towards
proline early in development [J. L. Purdy and S. C. Bondy,
Neuroscience 1, 125 (1976)], plasma levels may not be adequately
describing brain levels, although previous analysis of a
CD-1.times.Pro/Re F3 cross revealed an increase in the brain
proline levels as well [C. F. Baxter, R. A. Baldwin, J. L. Davis,
J. J. Flood, Pharmac. Biochem. & Behav. 22, 1053 (1985)]. The
normal concentration of proline in human plasma is between 100 and
450 mM. The concentration of proline in cerebrospinal fluid is
considerably lower (4.2 mM) [C. R. Scriver and L. E. Rosenberg,
Aminoacid metabolism and its disorders (Saunders, Philadelphia,
1973)].
[0607] The nucleotide sequence obtained from the cerebellar cDNA
clones overlapped partly with the sequence of the kidney cDNA
clones (data not shown). Additional sequence was obtained using a
5'RACE approach on total human brain mRNA. As part of a D.
melanogaster/human sequencing project, Campbell and colleagues [H.
D. Campbell, G. C. Webb, I. G. Young, Hum. Genet. 101, 69 (1997)]
also identified a human cDNA with homology to slgA (the gene
product was assigned the name PRODH). The reported sequence is
identical to the one determined in this study with the exception of
a G insertion in the 3' UTR, 1793 base pairs downstream of the
initiator methionine. Analysis in a small number of individuals
suggests that this variation represents either a sequencing
artifact or a very rare polymorphism. In addition, homologues from
(intentionally left blank) C. elegans [GeneBank Z81060, Sulston et
al., Nature 356, 37 (1992)] and Arabidopsis thaliana ]N.
Verbruggen, X. -J. Hua, M. May, M. Montagu, Proc. Natl. Acad. Sci.
U.S.A. 93, 8787 (1996)] have also been reported.
Testing of Prodh-Deficient Mice and Wild Type Littermates
[0608] Forty two homozygous and 26 wild type littermate mice of
both sexes, 2-3 months old at the onset of testing were used for
the behavioral analysis. Animals were housed in groups of 2-4 and
were maintained on a reverse 12:12 h light-dark cycle with lights
off at 0700 hrs. All testing occurred between 0900 and 1800 hrs.
Prior to all testing, animals were handled and weighed. The
behavioral tests were performed in the following order: locomotor
activity, light/dark transition, PPI and habituation of the startle
response. At the conclusion of all behavioral assays the genotypes
of all mice were reconfirmed by PCR analysis.
[0609] Spontaneous locomotor activity was tested in an open field
apparatus, a clear acrylic chamber (40.5.times.40.5.times.30 cm)
equipped with infrared sensors for the automatic recording of
horizontal activity (Digiscan Model RXYZCM, Accuscan Instruments,
Inc., Columbus, Ohio). Each subject was initially placed in the
center of the chamber and total distance traveled over the next 15
min was used as the measure of activity. To minimize the influence
of anxiety on activity level, activity was monitored under
indirect, very dim light and sound-attenuated conditions.
[0610] Following the initial test of locomotor activity, the effect
of the mProdh mutation on anxiety-like behaviors (collectively
termed anxiety reactivity or emotionality) was recorded in a
dark/light exploratory model in a 2-compartment light/dark box. The
apparatus and conditions were similar to those used for the
locomotor assay, except that an enclosed black acrylic box
(40.times.20.5.times.20.5 cm) was inserted into the right half of
the chamber with an opening (13.times.5 cm) allowing for free
passage between the two compartments monitored by an infrared beam.
The open compartment was now directly illuminated by a 60-watt bulb
placed 60 cm above the floor of the compartment. Animals were
initially placed in the dark compartment and data collection
commenced immediately for 10 min. Previous work assessing the
effects of anxiolytic and anxiogenic agents has established the
validity of this behavioral procedure in evaluating anxiety-like
behaviors in rodents. Variables recorded as a measure of anxiety
included latency to emerge from the dark compartment into the more
aversive brightly lit compartment, and amount of time spent
ambulating in each of the two compartments.
[0611] Testing was conducted in a SR-Lab system (San Diego
Instruments, San Diego, Calif.). Each of two acoustically insulated
startle chambers contained a transparent acrylic cylinder (4 cm in
diameter) mounted on a frame to which a motion sensor was attached
for the detection and transduction of movement, and a sound
generation system for the delivery of background white noise and
acoustic stimuli. To ensure comparable sensitivities of these
detectors across the two chambers, a vibrating standardization unit
(San Diego Instruments, San Diego, Calif.) was used. A CompuAdd 386
microprocessor and SDI interface board and software were used for
the delivery of stimuli and response recording (100 1-ms readings
beginning at startle stimulus onset). Response amplitude was
calculated as the maximum response level occurring during the 100
ms recording. Both chambers were calibrated for equivalent stimulus
intensities and response sensitivities with a digital sound level
meter (RadioShack), and experimental groups were balanced across
chambers. Because animals can in principle habituate to the
prepulse [J. C. Gewirtz and M. Davis, Behav. Neurosci. 109, 388
(1995)], as well as to the startle stimulus [T. D. Blumenthal,
Psychophysiology 34, 446 (1997)], the number of trials was kept to
the essential minimum. The choice of the two prepulse bursts used
in this experiment was based on a pilot experiment. Immediately
after placement in the chamber, the animal was given a 5 min
acclimation period during which background noise (67 dB) was
continually present, and then received 6 sets of the following 7
trial types counterbalanced to control for order: Trial 1: 40 ms,
100 dB noise burst alone; Trial 2: 40 ms, 115 dB noise burst alone;
Trial 3-6: 100 dB or 115 dB startle stimuli preceded 100 ms by a 20
ms, 82 dB or 90 dB noise burst (prepulse); Trial 7:
no-stimulus/background noise alone (67 dB). Intertrial interval was
variable (10-20 sec with an average of 15 sec). At the beginning of
the block of 42 trials, the animal received the following 3 trials:
1 no-stimulus/background noise alone (67 dB) trial, 1 startle
stimulus alone trial for both 100 dB and 115 dB. At the end of the
block of 42 trials the animal received the same 3 trials again in
reverse order. The background noise level was 67 dB during the
entire testing session. Data were analyzed using ANOVA with
repeated measures.
[0612] In order to obtain additional information on the perception
of the employed prepulse stimuli by wild type and mutant mice, one
week after the completion of the PPI experiment, prepulse alone
startle responses were evaluated at 82 dB and 90 dB. Mice were
exposed to three consecutive 40 ms stimuli: 67 dB white noise
(background trial), 82 dB (prepulse 1 trial), and 90 dB (prepulse 2
trial), distributed pseudorandomly and separated by an average of
15 sec intertrial intervals. This sequence was repeated 10 times,
that is, each stimulus were presented 30 times. The background
noise level was 67 dB during the entire testing session. Whereas a
prepulse alone startle response to 82 dB was barely detectable over
background for both genotypes, a response to 90 dB could be
reliably evaluated [there was a significant difference between
response levels to 67 dB and 90 dB for both wild type (p=0.0017)
and homozygous mutant animals (p=0.0001)]. The amplitude of the
startle response at 90 dB was .sup..about.20% of the one observed
for the 100 dB startle stimulus and it was indistinguishable
between the two genotypes (p=0.6). Induction of a startle response
by the 90 dB prepulse stimulus is unlikely to interfere with the
interpretation of the observed PPI patterns since previous
extensive analysis of different mouse strains demonstrated that no
correlation exists between the startle response to 90 dB stimulus
and the level of prepulse inhibition obtained with a 90 dB
prepulse. Moreover, the same analysis showed that there is no
correlation between the strain-specific dB threshold for induction
of a startle response and the level for prepulse inhibition with
any of the prepulse stimuli.
[0613] Habituation of the acoustic startle response was evaluated
as follows: 36 homozygous mutant and 20 wild type animals were
exposed to startle stimuli of broadband 115 dB for 40 ms with an
average of 15 sec (10-20 sec) intertrial interval, in the presence
of 67 dB broadband background noise. The session began with 5 min
acclimation period, followed by 5 consecutive administrations of
the 40 ms broad-band 115 dB burst. This block was repeated 24
times, resulting to 120 consecutive presentations of the 115 dB
noise. Weight-corrected startle magnitude values (Vmax) were
averaged for each block, expressed as percentage of the first block
and analyzed using ANOVA with repeated measures. In addition,
slopes of the habituation curves were analyzed by simple linear
regression analysis.
Results and Conclusion
[0614] Prodh-deficient mice and wild type littermates were examined
for abnormalities in sensorimotor gating. PPI was recorded, using a
combination of two startle levels and two prepulse levels and
expressed as 100-[(response to startle stimulus following
pre-pulse/response to startle stimulus alone).times.100] (such that
higher percentages represent greater levels of inhibition, FIG.
3A). This experiment demonstrated a significant attenuation in the
overall level of PPI in the homozygous mutant mice compared to wild
type littermates [F(1,66)=6.14, p=0.015] (FIG. 3B). No sex
differences were noted (p=0.866) and the % PPI increased with
increasing prepulse levels in both genotypes. Two features of the
startle responses were also evaluated: amplitude of the startle
response (ASR) and habituation (the decrement in the ASR over
repeated presentations of the same stimulus, a measure of
plasticity exhibited by startle responses). ASR can be affected by
several factors, including the integrity and excitability of the
responding neurons in subcortical auditory centers [J. F. Willot,
J. Kulig, T. Satterfield, Hear. Res. 16, 161 (1984); M. Koch, C.
Kling, C. M. Becker, Neuroreport 7, 806, 1996]. Analysis by
genotype did not reveal significant differences in the
weight-corrected startle amplitudes between wild type and
Prodh-deficient mice (FIG. 3C). Although a number of studies seem
to suggest a dissociation between startle amplitude and PPI [R.
Paylor and J. N. Crawley, Psychopharmacology 132, 169 (1997); V. P.
Bakshi, N. R. Swerdlow, M. A. Geyer, J. Pharmacol. Exp. Ther. 271,
787 (1994); C. Johansson, D. M. Jackson, J. Zhang, L. Svensson,
Pharmacol. Biochem. Behav. 52, 649 (1995)], this result indicates
that the observed attenuation of PPI is not associated with an
impaired startle response and in addition reveals an anatomical and
functional integrity of the responsive neurons in the
PRODH-deficient mice. Furthermore, homozygous mutant mice did not
demonstrate any impairment in the habituation of the acoustic
startle, at least under the conditions tested (FIG. 3D) [C. J.
Wilson and P. M. Groves, J. Comp. & Physiol. Psychiatry 83, 492
(1973); M. A. Geyer and D. L. Braff, Psychophysiology 19, 1
(1982)]. Locomotor activity and anxiety (reactivity) were also
assayed. In sharp contrast to the observed sensorimotor gating
deficits, no significant differences in the total distance
traveled, stereotypic behavior, or time spent in margin versus
center (an estimate of anxiety) were observed among animals of
either genotype in the open field locomotor assay (FIG. 4A) [W. E.
Crusio, H. Schwegler, J. H. van Abeelen, Behav. Brain Res. 32, 80
(1989)]. In addition, no significant effect of genotype on latency
to emerge into the more aversive brightly lit compartment, or time
spent ambulating in either light or dark compartment was observed
in the dark-light assay for anxiety (FIG. 4B) [J. Crawley and F. K.
Goodwin, Pharmacol. Biochem. Behav. 13, 167 (1980); C. Mathis, S.
M. Paul, J. N. Crawley, Behav. Genet. 24, 171 (1994); C. Mathis, P.
E. Neumann, H. Gershenfeld, S. M. Paul, J. N. Crawley, Behav.
Genet. 25, 557 (1995)].
[0615] Elevated proline concentration has been previously
associated with behavioral deficits in D. melanogaster (defective
phototaxis and low locomotor activity with indecisive movement
pattern). The marked behavioral abnormalities observed in the slgA
mutant flies correlated with a twofold elevation of proline in the
brain [D. C. Hayward et al., Proc. Natl. Acad. Sci. U.S.A. 90, 2979
(1993)], suggesting that behavioral effects may not necessarily
depend on grossly elevated proline levels. In humans, PRODH
deficiency is likely to be associated with hyperprolinemia type I,
a rare metabolic disorder [J. M. Phang, G. C. Yeh, C. R. Scriver,
in The Metabolic and Molecular Bases of Inherited Disease, C. R.
Scriver, A. L. Beaudet, W. S. Sly, D. Valle, Eds. (McGraw-Hill,
N.Y., 1995), pp. 1125-1146). Unlike most metabolic phenotypes,
hyperprolinemia type I heterozygotes (such as patients with
hemizygous 22q11 microdeletions) can demonstrate intermediate level
increases of plasma proline [J. Jacken, N. Goemans, J. -P. Fryns,
I. Francois, F. de Zegher, J. Inherit. Metab. Dis. 19, 275 (1996);
Phang, et al., (1995)]. The nature of the effects of abnormally
high proline (as in hyperprolinemia type I homozygotes) remain
controversial and an exact link between the defect in proline
metabolism and a behavioral/neurological phenotype in humans is
still lacking. Several of the initially proposed features of
hyperprolinemia type I (such as learning deficits) have not been
observed reproducibly even among hyperprolinemic siblings of
affected probands [F. Mollica and L. Provone, Acta Paediatr. Scand.
65, 206 (1976)]. Although under no obligation to explain such
results, and certainly not intending to be bound by any hypothesis
to explain these results, it is possible that fortuitous clinical
findings, allelic heterogeneity and genetic background-dependent
penetrance account for the variable observations. The rarity of
this disorder and the phenotypic inconsistencies have impeded
systematic psychiatric evaluation of these patients, although three
cases of patients with psychiatric symptoms (schizophrenia,
psychotic symptoms, autism) and increased blood proline levels have
been reported [T. L. Perry, J. W. Wright, S. Hansen, Biol.
Psychiatry 18, 89 (1983); M. Efron, N. Engl. J. Med. 272, 1243
(1965); T. Rokkones and A. C. L.o slashed.ken, Acta Paediatr.
Scand. 57, 225 (1968)]. In mice, initial analysis of the original
Pro/Re strain, revealed specific learning deficits J. L. Davis, R.
M. Pico, J. F. Flood, Behav. Neur. Biol. 48,128 (1987)] and studies
in chicks have shown that intracerebral administration of proline
may result in selective disruption of memory formation [A. Cherkin,
M. J. Eckardt, L. K. Gerbrandt, Science 193, 242 (1976)].
Identification and Characterization of Variant Alleles of the Human
Proline Dehydrogenase Gene
[0616] In the second study reported herein, common sequence
variations in the more conserved C-terminal part of the human cDNA
were scanned in ninety-two individuals affected with schizophrenia.
Genomic PCR and primarily reverse transcription (RT) PCR using
total mRNA isolated from transformed lymphocytes was applied. Eight
polymorphisms were identified in this region:
[0617] a silent G to A transition at the third position of codon
83, which introduces a PstI site;
[0618] a C to T transition at the first position of codon 101 which
results in a substitution of arginine for tryptophan;
[0619] a G to A transition at the second position of codon 101
which results in a substitution of arginine for glutamine;
[0620] a silent C to T transition at the first position of codon
247;
[0621] a C to T transition in the third position of codon 342;
[0622] a C to T transition in the third position of codon 421;
[0623] an A to G transition at the second position of codon 437,
which results in a substitution of glycine for arginine; and
[0624] a silent T to C transition at the first position of codon
497, which introduces a PvuII site.
[0625] Experiments using RT-PCR with primers flanking introns,
showed that all variations were present in PRODH mRNA. Analysis of
these variations in more extended samples revealed that the PvuII
polymorphism is relatively frequent in that .sup.-30% of
chromosomes in a sample of patients with schizophrenia displayed
this variation.
[0626] Moreover, statistical data from particular families whose
members suffer from schizophrenia have been gathered, and
demonstrate a direct link between the PRODH gene polymorphism
comprising the silent T to C transition at the first position of
codon 497, which introduces a PvuII site, and susceptibility of a
subject to schizophrenia. In particular, a transmission
disequilibrium test (TDT) [Spielman, R. S. et al. Transmission test
for linkage disequilibirium: the insulin gene region and
insulin-dependent diabetes mellitus (IDDM). Am. J. Hum. Genet.
(1993), 53:506-516] was performed on data from families in which
members suffered from schizophrenia. This test takes affected
individuals with their two parents. All three are typed for a
marker, one allele of which is believed to be associated with the
disease. The TDT considers the cases where at least one parent is
heterozygous at the marker allele (M1) which is suspected of being
associated with the disease. One of the two marker alleles of each
heterozygous patent is transmitted to each affected offspring and
one is not. The test compares the frequency of M1 among the
transmitted and nontransmitted alleles. The significance of the
association is tested by a simple X.sup.2 test (Table 1).
1TABLE 1 Tests to determine whether marker allele M1 is associated
with a disease. TDT test Nontransmitted allele Transmitted allele
M1 Not M1 M1 a b not M1 c d For either test, .chi..sup.2 (1 degree
of freedom) = (b - c).sup.2 / (b + c) The TDT: families are
selected where affected probands have at least one parent who is
heterozygous for M1. The transmitted and nontransmitted parental
alleles are compared.
[0627] The results of the TDT, set forth in FIG. 5, show there is a
statistically significant preferential transmission of the PRODH
gene polymorphism comprising the silent T to C transition at the
first position of codon 497, which introduces a PvuII site, and
susceptibility of a subject to schizophrenia, or disease or
disorders related thereto, such as obsessive compulsive disorder
(OCD), bipolar disorder (BP), or major depressive disorder
(MDD).
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[0714] The present invention is not to be limited in scope by the
specific embodiments describe herein. Indeed, various modifications
of the invention in addition to those described herein will become
apparent to those skilled in the art from the foregoing description
and the accompanying figures. Such modifications are intended to
fall within the scope of the appended claims.
[0715] It is further to be understood that all base sizes or amino
acid sizes, and all molecular weight or molecular mass values,
given for nucleic acids or polypeptides are approximate, and are
provided for description.
[0716] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties.
Sequence CWU 1
1
9 1 1551 DNA Homo sapiens 1 atgctggaat ttgtgatgag agagtggaaa
aaatccagga aacttctagg acagaggcta 60 ttcaacaagc tcatgaagat
gaccttctat gggcattttg tagccgggga ggaccaggag 120 tccatccagc
ccctgcttcg gcactacagg gccttcggtg tcagcgccat cctggactat 180
ggagtggagg aggacctgag ccccgaggag gcagagcaca aggagatgga gtcctgcacc
240 tcagctgcgg agagggatgg cagtggcacg aataagcggg acaagcaata
ccaggcccac 300 cgggccttcg gggaccgcag gaatggtgtc atcagtgccc
gcacctactt ctacgccaat 360 gaggccaagt gcgacagcca catggagaca
ttcttgcgct gcatcgaagc ctcaggtaga 420 gtcagcgatg acggcttcat
agccattaag ctcacagcac tggggagacc ccagtttctg 480 ctgcagttct
cagaggtgct ggccaagtgg aggtgcttct ttcaccaaat ggctgtggag 540
caagggcagg cgggcctggc tgccatggac accaagctgg aggtggcggt gctgcaggaa
600 agtgtcgcaa agttgggcat cgcatccagg gctgagattg aggactggtt
cacggcagag 660 accctgggag tgtctggcac catggacctg ctggactgga
gcagcctcat cgacagcagg 720 accaagctgt ccaagcacct ggtagtcccc
aacgcacaga caggacagct ggagcccctg 780 ctgtcccggt tcactgagga
ggaggagcta cagatgacca ggatgctaca gcggatggat 840 gtcctggcca
agaaagccac agagatgggc gtgcggctga tggtggatgc cgagcagacc 900
tacttccagc cggccatcag ccgcctgacg ctggagatgc agcggaagtt caatgtggag
960 aagccgctca tcttcaacac ataccagtgc tacctcaagg atgcctatga
caatgtgacc 1020 ctggacgtgg agctggctcg ccgtgagggc tggtgttttg
gggccaagct ggtgcggggc 1080 gcatacctgg cccaggagcg agcccgtgcg
gcagagatcg gctatgagga ccccatcaac 1140 cccacgtacg aggccaccaa
cgccatgtac cacaggtgcc tggactacgt gttggaggag 1200 ctgaagcaca
acgccaaggc caaggtgatg gtggcctccc acaatgagga cacagtgcgc 1260
ttcgcactgc gcaggatgga ggagctgggc ctgcatcctg ctgaccacca ggtgtacttt
1320 ggacagctgc taggcatgtg tgaccagatc agcttcccgc tgggccaggc
cggctacccc 1380 gtgtacaagt acgtgcccta tggccccgtg atggaggtgc
tgccctactt gtcccgccgt 1440 gccctggaga acagcagcct catgaagggc
acccatcggg agcggcagtt gctgtggctg 1500 gagctcttga ggcggctccg
aactggcaac ctcttccatc gccctgccta g 1551 2 516 PRT Homo sapiens 2
Met Leu Glu Phe Val Met Arg Glu Trp Lys Lys Ser Arg Lys Leu Leu 1 5
10 15 Gly Gln Arg Leu Phe Asn Lys Leu Met Lys Met Thr Phe Tyr Gly
His 20 25 30 Phe Val Ala Gly Glu Asp Gln Glu Ser Ile Gln Pro Leu
Leu Arg His 35 40 45 Tyr Arg Ala Phe Gly Val Ser Ala Ile Leu Asp
Tyr Gly Val Glu Glu 50 55 60 Asp Leu Ser Pro Glu Glu Ala Glu His
Lys Glu Met Glu Ser Cys Thr 65 70 75 80 Ser Ala Ala Glu Arg Asp Gly
Ser Gly Thr Asn Lys Arg Asp Lys Gln 85 90 95 Tyr Gln Ala His Arg
Ala Phe Gly Asp Arg Arg Asn Gly Val Ile Ser 100 105 110 Ala Arg Thr
Tyr Phe Tyr Ala Asn Glu Ala Lys Cys Asp Ser His Met 115 120 125 Glu
Thr Phe Leu Arg Cys Ile Glu Ala Ser Gly Arg Val Ser Asp Asp 130 135
140 Gly Phe Ile Ala Ile Lys Leu Thr Ala Leu Gly Arg Pro Gln Phe Leu
145 150 155 160 Leu Gln Phe Ser Glu Val Leu Ala Lys Trp Arg Cys Phe
Phe His Gln 165 170 175 Met Ala Val Glu Gln Gly Gln Ala Gly Leu Ala
Ala Met Asp Thr Lys 180 185 190 Leu Glu Val Ala Val Leu Gln Glu Ser
Val Ala Lys Leu Gly Ile Ala 195 200 205 Ser Arg Ala Glu Ile Glu Asp
Trp Phe Thr Ala Glu Thr Leu Gly Val 210 215 220 Ser Gly Thr Met Asp
Leu Leu Asp Trp Ser Ser Leu Ile Asp Ser Arg 225 230 235 240 Thr Lys
Leu Ser Lys His Leu Val Val Pro Asn Ala Gln Thr Gly Gln 245 250 255
Leu Glu Pro Leu Leu Ser Arg Phe Thr Glu Glu Glu Glu Leu Gln Met 260
265 270 Thr Arg Met Leu Gln Arg Met Asp Val Leu Ala Lys Lys Ala Thr
Glu 275 280 285 Met Gly Val Arg Leu Met Val Asp Ala Glu Gln Thr Tyr
Phe Gln Pro 290 295 300 Ala Ile Ser Arg Leu Thr Leu Glu Met Gln Arg
Lys Phe Asn Val Glu 305 310 315 320 Lys Pro Leu Ile Phe Asn Thr Tyr
Gln Cys Tyr Leu Lys Asp Ala Tyr 325 330 335 Asp Asn Val Thr Leu Asp
Val Glu Leu Ala Arg Arg Glu Gly Trp Cys 340 345 350 Phe Gly Ala Lys
Leu Val Arg Gly Ala Tyr Leu Ala Gln Glu Arg Ala 355 360 365 Arg Ala
Ala Glu Ile Gly Tyr Glu Asp Pro Ile Asn Pro Thr Tyr Glu 370 375 380
Ala Thr Asn Ala Met Tyr His Arg Cys Leu Asp Tyr Val Leu Glu Glu 385
390 395 400 Leu Lys His Asn Ala Lys Ala Lys Val Met Val Ala Ser His
Asn Glu 405 410 415 Asp Thr Val Arg Phe Ala Leu Arg Arg Met Glu Glu
Leu Gly Leu His 420 425 430 Pro Ala Asp His Gln Val Tyr Phe Gly Gln
Leu Leu Gly Met Cys Asp 435 440 445 Gln Ile Ser Phe Pro Leu Gly Gln
Ala Gly Tyr Pro Val Tyr Lys Tyr 450 455 460 Val Pro Tyr Gly Pro Val
Met Glu Val Leu Pro Tyr Leu Ser Arg Arg 465 470 475 480 Ala Leu Glu
Asn Ser Ser Leu Met Lys Gly Thr His Arg Glu Arg Gln 485 490 495 Leu
Leu Trp Leu Glu Leu Leu Arg Arg Leu Arg Thr Gly Asn Leu Phe 500 505
510 His Arg Pro Ala 515 3 1494 DNA Mus musculus 3 atgttcgaga
gattgatgaa gatgaccttc tatggccatt ttgtggctgg cgaagaccag 60
gagtctatca ggcctctgat ccggcacaac aaagcctttg gtgttggctt tatcctggac
120 tatggagtgg aggaagatct gagccctgag gaggcggagc gcaaagagat
ggagtcatgc 180 acttctgaag cagagagaga tggcagtgga gcaaataaga
gggagaagca gtatcaggtg 240 caccccgcct ttggagaccg cagagatggt
gtcatcagtg cccgcaccta cttctatgcc 300 aatgaagcca agtgtgacaa
ctacatggag aacttactgc agtgcatcaa ggcctcaggt 360 ggagccagtg
atggtggttt ctcagccatt aagctcactg cactggggag accacagttt 420
ctgctgcagt tctcagacgt gctgaccagg tggagacggt tcttccatca aatggctgca
480 gagcagggac aggctgggcg tgctgctgta gacacaaagc tggaggtggc
ggtgctccag 540 gacagcatcg caaagatggg catcgcatcc agggctgaga
ttgaagggtg gttcacgcca 600 gagacgctgg gagtgtctgg caccgtggac
ttgctggact ggaacagcct cattgacagc 660 aggacccggc tctccaggca
cttggtggtc cccaatgtgc agactggcca gctggagccc 720 ctgctgtcac
ggttcactga ggaggaagag cagcagatga aaaggatgct gcagaggatg 780
gatgtactgg ccaagaaagc aaaagaagca ggtgtgcgcc tgatgattga tgctgagcag
840 agctacttcc aaccagccat cagccgcctg accctggaga tgcagcgcag
gttcaatgtg 900 gataagccgt tcatcttcaa cacattccag tgctacctca
aggatgccta tgacaatgtg 960 accttggata tggaactggc tcgccgtgag
ggctggtgtt ccggggccaa gctggtacgt 1020 cgtgcataca tggcccaaga
gcgtgtcagg gcagcagaga tcggttatga agaccccatc 1080 aaccctacat
atgaagccac caatgctatg taccacaggt gccttaacta tgttctggag 1140
gagctgaagc acagcaccaa ggcagaagtg atggtggctt cccacaacga ggacaccgtg
1200 cacttcackt tgtgcaggat gaaggagata ggcctgcatc ctgctgatgg
tcaggtgtgc 1260 ttcggacagc tgctggggat gtgtgaccaa atcagcttcc
cactaggcca ggcaggcttt 1320 cctgtgtaca agtatgtgcc ctatggccct
gtgatggagg tactccctta cctgtcccgc 1380 cgtgccctgg agaacagcag
catcatgaag ggtgctcagc gagagaggca gctgctatgg 1440 caggagctcc
gcaggcggct gcgcactggc agcctcttcc accatccggc ctag 1494 4 497 PRT Mus
musculus 4 Met Phe Glu Arg Leu Met Lys Met Thr Phe Tyr Gly His Phe
Val Ala 1 5 10 15 Gly Glu Asp Gln Glu Ser Ile Arg Pro Leu Ile Arg
His Asn Lys Ala 20 25 30 Phe Gly Val Gly Phe Ile Leu Asp Tyr Gly
Val Glu Glu Asp Leu Ser 35 40 45 Pro Glu Glu Ala Glu Arg Lys Glu
Met Glu Ser Cys Thr Ser Glu Ala 50 55 60 Glu Arg Asp Gly Ser Gly
Ala Asn Lys Arg Glu Lys Gln Tyr Gln Val 65 70 75 80 His Pro Ala Phe
Gly Asp Arg Arg Asp Gly Val Ile Ser Ala Arg Thr 85 90 95 Tyr Phe
Tyr Ala Asn Glu Ala Lys Cys Asp Asn Tyr Met Glu Asn Leu 100 105 110
Leu Gln Cys Ile Lys Ala Ser Gly Gly Ala Ser Asp Gly Gly Phe Ser 115
120 125 Ala Ile Lys Leu Thr Ala Leu Gly Arg Pro Gln Phe Leu Leu Gln
Phe 130 135 140 Ser Asp Val Leu Thr Arg Trp Arg Arg Phe Phe His Gln
Met Ala Ala 145 150 155 160 Glu Gln Gly Gln Ala Gly Arg Ala Ala Val
Asp Thr Lys Leu Glu Val 165 170 175 Ala Val Leu Gln Asp Ser Ile Ala
Lys Met Gly Ile Ala Ser Arg Ala 180 185 190 Glu Ile Glu Gly Trp Phe
Thr Pro Glu Thr Leu Gly Val Ser Gly Thr 195 200 205 Val Asp Leu Leu
Asp Trp Asn Ser Leu Ile Asp Ser Arg Thr Arg Leu 210 215 220 Ser Arg
His Leu Val Val Pro Asn Val Gln Thr Gly Gln Leu Glu Pro 225 230 235
240 Leu Leu Ser Arg Phe Thr Glu Glu Glu Glu Gln Gln Met Lys Arg Met
245 250 255 Leu Gln Arg Met Asp Val Leu Ala Lys Lys Ala Lys Glu Ala
Gly Val 260 265 270 Arg Leu Met Ile Asp Ala Glu Gln Ser Tyr Phe Gln
Pro Ala Ile Ser 275 280 285 Arg Leu Thr Leu Glu Met Gln Arg Arg Phe
Asn Val Asp Lys Pro Phe 290 295 300 Ile Phe Asn Thr Phe Gln Cys Tyr
Leu Lys Asp Ala Tyr Asp Asn Val 305 310 315 320 Thr Leu Asp Met Glu
Leu Ala Arg Arg Glu Gly Trp Cys Ser Gly Ala 325 330 335 Lys Leu Val
Arg Arg Ala Tyr Met Ala Gln Glu Arg Val Arg Ala Ala 340 345 350 Glu
Ile Gly Tyr Glu Asp Pro Ile Asn Pro Thr Tyr Glu Ala Thr Asn 355 360
365 Ala Met Tyr His Arg Cys Leu Asn Tyr Val Leu Glu Glu Leu Lys His
370 375 380 Ser Thr Lys Ala Glu Val Met Val Ala Ser His Asn Glu Asp
Thr Val 385 390 395 400 His Phe Thr Leu Cys Arg Met Lys Glu Ile Gly
Leu His Pro Ala Asp 405 410 415 Gly Gln Val Cys Phe Gly Gln Leu Leu
Gly Met Cys Asp Gln Ile Ser 420 425 430 Phe Pro Leu Gly Gln Ala Gly
Phe Pro Val Tyr Lys Tyr Val Pro Tyr 435 440 445 Gly Pro Val Met Glu
Val Leu Pro Tyr Leu Ser Arg Arg Ala Leu Glu 450 455 460 Asn Ser Ser
Ile Met Lys Gly Ala Gln Arg Glu Arg Gln Leu Leu Trp 465 470 475 480
Gln Glu Leu Arg Arg Arg Leu Arg Thr Gly Ser Leu Phe His His Pro 485
490 495 Ala 5 20 DNA Artificial Sequence Description of Artificial
SequencePRIMER 5 gaccaaatca gcttcccact 20 6 20 DNA Artificial
Sequence 6 cccttcatga tgctgctgtt 20 7 2240 DNA Mus musculus 7
agcgcgtctt cttgctgcgg tcggtggcac cacgcgtcgc tgccctctca accaaaccgc
60 aagcccagga acagcctccc gcgagccctg aggctcttcg gggatgtggg
gcggccaagg 120 ctgtgcggcc gcctgtgcca gccgtggact tcaccaacac
gcaggaggcg tatcgcagcc 180 ggcggagttg ggagttggtg cgcaacctgc
tagtgctgcg gctgtgtgcg tcgccggtgc 240 tgctagcgca ccacgagcag
ttgttccaag ttgccaggaa gcttctgggg caaaggatgt 300 tcgagagatt
gatgaagatg accttctatg gccattttgt ggctggcgaa gaccaggagt 360
ctatcaggcc tctgatccgg cacaacaaag cctttggtgt tggctttatc ctggactatg
420 gagtggagga agatctgagc cctgaggagg cggagcgcaa agagatggag
tcatgcactt 480 ctgaagcaga gagagatggc agtggagcaa ataagaggga
gaagcagtat caggtgcacc 540 ccgcctttgg agaccgcaga gatggtgtca
tcagtgcccg cacctacttc tatgccaatg 600 aagccaagtg tgacaactac
atggagaact tactgcagtg catcaaggcc tcaggtggag 660 ccagtgatgg
tggtttctca gccattaagc tcactgcact ggggagacca cagtttctgc 720
tgcagttctc agacgtgctg accaggtgga gacggttctt ccatcaaatg gctgcagagc
780 agggacaggc tgggcgtgct gctgtagaca caaagctgga ggtggcggtg
ctccaggaca 840 gcatcgcaaa gatgggcatc gcatccaggg ctgagattga
agggtggttc acgccagaga 900 cgctgggagt gtctggcacc gtggacttgc
tggactggaa cagcctcatt gacagcagga 960 cccggctctc caggcacttg
gtggtcccca atgtgcagac tggccagctg gagcccctgc 1020 tgtcacggtt
cactgaggag gaagagcagc agatgaaaag gatgctgcag aggatggatg 1080
tactggccaa gaaagcaaaa gaagcaggtg tgcgcctgat gattgatgct gagcagagct
1140 acttccaacc agccatcagc cgcctgaccc tggagatgca gcgcaggttc
aatgtggata 1200 agccgttcat cttcaacaca ttccagtgct acctcaagga
tgcctatgac aatgtgacct 1260 tggatatgga actggctcgc cgtgagggct
ggtgttccgg ggccaagctg gtacgtcgtg 1320 catacatggc ccaagagcgt
gtcagggcag cagagatcgg ttatgaagac cccatcaacc 1380 ctacatatga
agccaccaat gctatgtacc acaggtgcct taactatgtt ctggaggagc 1440
tgaagcacag caccaaggca gaagtgatgg tggcttccca caacgaggac accgtgcact
1500 tcackttgtg caggatgaag gagataggcc tgcatcctgc tgatggtcag
gtgtgcttcg 1560 gacagctgct ggggatgtgt gaccaaatca gcttcccact
aggccaggca ggctttcctg 1620 tgtacaagta tgtgccctat ggccctgtga
tgtaggtact cccttacctg tcccgccgtg 1680 ccctggagaa cagcagcatc
atgaagggtg ctcagcgaga gaggcagctg ctatggcagg 1740 agctccgcag
gcggctgcgc actggcagcc tcttccacca tccggcctag tcaccgcagg 1800
agccttgccc acccgctcgt actccactca accccttacc tctggggctt caggcggggc
1860 acagcttggg attgggctgg ggttccttaa cccaacctgc ccagacacag
ttcacctttt 1920 tatgcccaag gctttttatg cccaaggcgg gatttcatca
gtggacagtt cctgaggaac 1980 agtgcccaag atggtcgtct ggtcacagag
gctgccttct gggacttcct gtaccccaag 2040 gaacagacac tcaggagtgg
ggtcagttag agcccctggg agctgcccca ctaatttgag 2100 taagcactga
ccacttctgc aggttacaga gccctagtcc aggattaacc ttctgccagg 2160
gtctaaccca ttttccctgc actgggcaga ggacagacta ggaagcctgt ttagtcaata
2220 aatcatcctg taacagagtc 2240 8 452 PRT Mus musculus 8 Met Phe
Glu Arg Leu Met Lys Met Thr Phe Tyr Gly His Phe Val Ala 1 5 10 15
Gly Glu Asp Gln Glu Ser Ile Arg Pro Leu Ile Arg His Asn Lys Ala 20
25 30 Phe Gly Val Gly Phe Ile Leu Asp Tyr Gly Val Glu Glu Asp Leu
Ser 35 40 45 Pro Glu Glu Ala Glu Arg Lys Glu Met Glu Ser Cys Thr
Ser Glu Ala 50 55 60 Glu Arg Asp Gly Ser Gly Ala Asn Lys Arg Glu
Lys Gln Tyr Gln Val 65 70 75 80 His Pro Ala Phe Gly Asp Arg Arg Asp
Gly Val Ile Ser Ala Arg Thr 85 90 95 Tyr Phe Tyr Ala Asn Glu Ala
Lys Cys Asp Asn Tyr Met Glu Asn Leu 100 105 110 Leu Gln Cys Ile Lys
Ala Ser Gly Gly Ala Ser Asp Gly Gly Phe Ser 115 120 125 Ala Ile Lys
Leu Thr Ala Leu Gly Arg Pro Gln Phe Leu Leu Gln Phe 130 135 140 Ser
Asp Val Leu Thr Arg Trp Arg Arg Phe Phe His Gln Met Ala Ala 145 150
155 160 Glu Gln Gly Gln Ala Gly Arg Ala Ala Val Asp Thr Lys Leu Glu
Val 165 170 175 Ala Val Leu Gln Asp Ser Ile Ala Lys Met Gly Ile Ala
Ser Arg Ala 180 185 190 Glu Ile Glu Gly Trp Phe Thr Pro Glu Thr Leu
Gly Val Ser Gly Thr 195 200 205 Val Asp Leu Leu Asp Trp Asn Ser Leu
Ile Asp Ser Arg Thr Arg Leu 210 215 220 Ser Arg His Leu Val Val Pro
Asn Val Gln Thr Gly Gln Leu Glu Pro 225 230 235 240 Leu Leu Ser Arg
Phe Thr Glu Glu Glu Glu Gln Gln Met Lys Arg Met 245 250 255 Leu Gln
Arg Met Asp Val Leu Ala Lys Lys Ala Lys Glu Ala Gly Val 260 265 270
Arg Leu Met Ile Asp Ala Glu Gln Ser Tyr Phe Gln Pro Ala Ile Ser 275
280 285 Arg Leu Thr Leu Glu Met Gln Arg Arg Phe Asn Val Asp Lys Pro
Phe 290 295 300 Ile Phe Asn Thr Phe Gln Cys Tyr Leu Lys Asp Ala Tyr
Asp Asn Val 305 310 315 320 Thr Leu Asp Met Glu Leu Ala Arg Arg Glu
Gly Trp Cys Ser Gly Ala 325 330 335 Lys Leu Val Arg Arg Ala Tyr Met
Ala Gln Glu Arg Val Arg Ala Ala 340 345 350 Glu Ile Gly Tyr Glu Asp
Pro Ile Asn Pro Thr Tyr Glu Ala Thr Asn 355 360 365 Ala Met Tyr His
Arg Cys Leu Asn Tyr Val Leu Glu Glu Leu Lys His 370 375 380 Ser Thr
Lys Ala Glu Val Met Val Ala Ser His Asn Glu Asp Thr Val 385 390 395
400 His Phe Thr Leu Cys Arg Met Lys Glu Ile Gly Leu His Pro Ala Asp
405 410 415 Gly Gln Val Cys Phe Gly Gln Leu Leu Gly Met Cys Asp Gln
Ile Ser 420 425 430 Phe Pro Leu Gly Gln Ala Gly Phe Pro Val Tyr Lys
Tyr Val Pro Tyr 435 440 445 Gly Pro Val Met 450 9 2389 DNA Homo
sapiens 9 taatgagagg gaaaacaagt atgaagctgt gtggctgaaa ccgtcttggc
aagttaggga 60 aagaaaacgg aagtcactgg ggctgatcac agtgctaagc
atgagagcac tgcaagatga 120 ggtcacggag gtgggcaggg accggcttgt
gccaggcctt gctggcaggg tgaagagttt 180 gccttttctc tgcgtacaat
ggaaaggaga agaggtttta agcaagagaa tggcttggtc 240 atgtgtatgt
ctttgagaca ccctggctag tctatgtatg atgcaaaagg tgggtggggc 300
agggtgacaa gaaaatactg ttccggagct tcctgtggct gtgcctataa
gaggtggtgg
360 tggtggtgtg gaaggaggtg tggcagtgaa taaacagaga tgtagaaaca
gcgtgtacat 420 atattttaag gaacactgag gacgtgatgc tggaatttgt
gatgagagag tggaaaaaat 480 ccaggaaact tctaggacag aggctattca
acaagctcat gaagatgacc ttctatgggc 540 attttgtagc cggggaggac
caggagtcca tccagcccct gcttcggcac tacagggcct 600 tcggtgtcag
cgccatcctg gactatggag tggaggagga cctgagcccc gaggaggcag 660
agcacaagga gatggagtcc tgcacctcag ctgcggagag ggatggcagt ggcacgaata
720 agcgggacaa gcaataccag gcccaccggg ccttcgggga ccgcaggaat
ggtgtcatca 780 gtgcccgcac ctacttctac gccaatgagg ccaagtgcga
cagccacatg gagacattct 840 tgcgctgcat cgaagcctca ggtagagtca
gcgatgacgg cttcatagcc attaagctca 900 cagcactggg gagaccccag
tttctgctgc agttctcaga ggtgctggcc aagtggaggt 960 gcttctttca
ccaaatggct gtggagcaag ggcaggcggg cctggctgcc atggacacca 1020
agctggaggt ggcggtgctg caggaaagtg tcgcaaagtt gggcatcgca tccagggctg
1080 agattgagga ctggttcacg gcagagaccc tgggagtgtc tggcaccatg
gacctgctgg 1140 actggagcag cctcatcgac agcaggacca agctgtccaa
gcacctggta gtccccaacg 1200 cacagacagg acagctggag cccctgctgt
cccggttcac tgaggaggag gagctacaga 1260 tgaccaggat gctacagcgg
atggatgtcc tggccaagaa agccacagag atgggcgtgc 1320 ggctgatggt
ggatgccgag cagacctact tccagccggc catcagccgc ctgacgctgg 1380
agatgcagcg gaagttcaat gtggagaagc cgctcatctt caacacatac cagtgctacc
1440 tcaaggatgc ctatgacaat gtgaccctgg acgtggagct ggctcgccgt
gagggctggt 1500 gttttggggc caagctggtg cggggcgcat acctggccca
ggagcgagcc cgtgcggcag 1560 agatcggcta tgaggacccc atcaacccca
cgtacgaggc caccaacgcc atgtaccaca 1620 ggtgcctgga ctacgtgttg
gaggagctga agcacaacgc caaggccaag gtgatggtgg 1680 cctcccacaa
tgaggacaca gtgcgcttcg cactgcgcag gatggaggag ctgggcctgc 1740
atcctgctga ccaccaggtg tactttggac agctgctagg catgtgtgac cagatcagct
1800 tcccgctggg ccaggccggc taccccgtgt acaagtacgt gccctatggc
cccgtgatgg 1860 aggtgctgcc ctacttgtcc cgccgtgccc tggagaacag
cagcctcatg aagggcaccc 1920 atcgggagcg gcagttgctg tggctggagc
tcttgaggcg gctccgaact ggcaacctct 1980 tccatcgccc tgcctagcac
ccgccagcac accctcagcc tccagcaccc cccgcccccg 2040 cccaggccat
caccacagct gcagccaacc ccatcctcac acagattcac cttttttcac 2100
cccacacttg cagagctgct ggaggtgagg tcaggtgcct cccagccctg cccagagtat
2160 gggcactcag gtgtgggccg aacctgatac ctgcctggga cagccactgg
aaacttttgg 2220 gaactctcct cgaatgtgtg gcccaaggcc cccacctctg
tgacccccat gtccttggac 2280 ctagaggatt gtccaccttc tgccaaggcc
agcccacaca gcccgagccc cttggggagc 2340 agtggccggg ctggggaggc
ctgcctggtc aataaaccac tgttcctgc 2389
* * * * *
References