U.S. patent application number 10/713810 was filed with the patent office on 2004-04-22 for methods and compositions for modulating neurodegeneration.
This patent application is currently assigned to California Institute of Technology, a California corporation. Invention is credited to Benzer, Seymour, Min, Kyung-Tai.
Application Number | 20040077581 10/713810 |
Document ID | / |
Family ID | 29714577 |
Filed Date | 2004-04-22 |
United States Patent
Application |
20040077581 |
Kind Code |
A1 |
Min, Kyung-Tai ; et
al. |
April 22, 2004 |
Methods and compositions for modulating neurodegeneration
Abstract
Disclosed are polypeptides and polynucleotides having very long
chain fatty acid coA synthetase activity. The polypeptides and
polynucleotides are useful in identifying and modulating
neurodegeneration.
Inventors: |
Min, Kyung-Tai; (Pasadena,
CA) ; Benzer, Seymour; (San Marino, CA) |
Correspondence
Address: |
FISH & RICHARDSON, PC
12390 EL CAMINO REAL
SAN DIEGO
CA
92130-2081
US
|
Assignee: |
California Institute of Technology,
a California corporation
|
Family ID: |
29714577 |
Appl. No.: |
10/713810 |
Filed: |
November 14, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10713810 |
Nov 14, 2003 |
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09418963 |
Oct 14, 1999 |
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6664039 |
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60104298 |
Oct 14, 1998 |
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Current U.S.
Class: |
514/44R ;
435/193; 435/320.1; 435/325; 435/6.16; 435/69.1; 536/23.2 |
Current CPC
Class: |
G01N 2500/00 20130101;
C12N 9/1029 20130101; Y10S 977/918 20130101; Y10S 977/915 20130101;
G01N 33/6896 20130101; C12Q 1/25 20130101 |
Class at
Publication: |
514/044 ;
435/006; 435/069.1; 435/193; 435/320.1; 435/325; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12P 021/02; C12N 005/06 |
Goverment Interests
[0002] The U.S. Government has certain rights in this invention
pursuant to Grant Nos. AG12289 and EY09278 awarded by the National
Institute of Health.
Claims
What is claimed is:
1. An isolated polynucleotide consisting of the nucleic acid
sequence set forth in SEQ ID NO:1.
2. An isolated polynucleotide comprising the nucleic acid sequence
set forth in SEQ ID NO:1.
3. An isolated polynucleotide encoding a polypeptide comprising the
amino acid sequence set forth in SEQ ID NO:2.
4. An isolated polynucleotide encoding a polypeptide the sequence
of which comprises the amino acid sequence of SEQ ID NO:2 with 0 to
50 conservative amino acid substitutions, wherein the polypeptide
is a very long chain fatty acid acyl (VLCFA) CoA synthetase that
converts a very long chain fatty acid to a thioester
derivative.
5. The isolated polynucleotide of claim 4, wherein the amino acid
sequence comprises 0 to 30 conservative amino acid
substitutions.
6. The isolated polynucleotide of claim 4, wherein the amino acid
sequence comprises 0 to 10 conservative amino acid
substitutions.
7. An isolated polynucleotide that hybridizes under stringent
conditions to a polynucleotide comprising the nucleic acid sequence
of SEQ ID NO:1, or complement thereof, wherein the polypeptide is a
very long chain fatty acid acyl (VLCFA) CoA synthetase that
converts a very long chain fatty acid to a thioester
derivative.
8. An isolated polynucleotide comprising a nucleotide sequence that
is at least 80% homologous to the nucleic acid sequence of SEQ ID
NO:1, wherein the polypeptide is a very long chain fatty acid acyl
(VLCFA) CoA synthetase that converts a very long chain fatty acid
to a thioester derivative.
9. The isolated polynucleotide of claim 8 comprising a nucleotide
sequence that is at least 90% homologous to the sequence of SEQ ID
NO:1.
10. The isolated polynucleotide of claim 8 comprising a nucleotide
sequence that is at least 95% homologous to the sequence of SEQ ID
NO:1.
11. An isolated polynucleotide comprising the nucleic acid sequence
set forth in SEQ ID NO:1, wherein thymine is uridine.
12. An isolated polynucleotide comprising a sequence that encodes a
polypeptide the amino acid sequence of which is at least 80%
identical to the sequence of SEQ ID NO:2.
13. The isolated polynucleotide of claim 12, wherein the amino acid
sequence is at least 90% identical to the sequence of SEQ ID
NO:2.
14. The isolated polynucleotide of claim 12, wherein the amino acid
sequence is at least 95% identical to the sequence of SEQ ID
NO:2
15. A vector comprising the polynucleotide of claim 1, 2, 3, 4, 7,
8, 11 or 12.
16. The vector of claim 12, wherein the vector is an expression
vector.
17. The vector of claim 12, wherein the vector is a plasmid.
18. The vector of claim 12, wherein the vector is a viral
vector.
19. A host cell transformed with the vector of claim 12.
20. The host cell of claim 16, wherein the cell is a eukaryotic
cell.
21. The host cell of claim 16, wherein the cell is a prokaryotic
cell.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. patent
application Ser. No. 09/418,963, filed Oct. 14, 1999, which claims
priority from Provisional Application Serial No. 60/104,298, filed
Oct. 14, 1998, each of which is incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0003] This invention relates to newly identified polynucleotides,
polypeptides encoded by such polynucleotides, the use of such
polynucleotides and polypeptides, as well as the production and
isolation of such polynucleotides and polypeptides. More
particularly, methods and compositions useful in modulating
neurodegeneration are provided.
BACKGROUND OF THE INVENTION
[0004] Drosophila, with its short generation time, highly evolved
nervous system, and amenability to genetic and molecular
techniques, may be a useful model system for understanding
neurodegenerative diseases and for development of methods for
prevention and treatment. For instance, the mutants, drop-dead
(Benzer, S., J. Am. Med. Assn. 218:1015-1022 (1971)), and swiss
cheese (Kretzschmar et al., Neuroscience, 17:7425-7432 (1997)) show
late-onset degeneration in the adult brain; spongecake and eggroll
(Min and Benzer, Curr. Biol., 7:885-888 (1997)) exhibit brain
degeneration patterns similar to those seen in human diseases.
[0005] Adrenoleukodystrophy (ALD) in humans is manifested by
gradual neurological deterioration with demyelination, blindness
and early death. In a milder form, referred to as
adrenomyeloneuropathy, which involves mutations in the same gene,
there is later onset, with progressive paraparesis and distal
axonopathy (Moser, H. W., Brain, 120:1485-1508 (1997)). The gene
associated with ALD, has been identified as a member of the
ATP-binding cassette (ABC) transmembrane transporter superfamily
and may be needed for the transport of very long chain fatty acid
acyl (VLCFA) CoA synthetase (Mosser et al., Nature, 361:726-730
(1993)) resulting in decreased affinity of VLCFA-CoA synthase. The
deficiency in activity of the synthetase, which normally
metabolizes the VLCFAs, causes elevated levels of hexacosanoic acid
(C26:0) in serum (Moser, supra). Transfer of the normal cDNA for
the ATP-binding cassette transmembrane proteins into ALD
fibroblasts can correct the C25 level (Cartier et al., Proc. Natl.
Acad. Sci. USA, 92:1674-1678 (1995)).
[0006] In X-linked human ALD and in three knockout mice of the ABC
transporter gene (Kobayashi et al., Biochem Biophys. Res. Commun.
232:631 (1997); Foiss-Peller et al., J. Neurosci. Res. 50:829
(1997)) there was no correlation between the amount of VLCFAs and
the severity of pathology. Some individuals with high VLCFAs
escaped the neurological defects and the knock out mice did not
show the pathology of ALD in spite of having elevated VLCFAs. These
observations suggest that excess VLCFAs and the pathology may not
have a direct causal relationship, but may be separate
ramifications of another, underlying defect.
SUMMARY OF THE INVENTION
[0007] In a first embodiment, the present invention provides a
substantially purified very long chain fatty acid coA-synthetase
(VLCFA coA-syn) polypeptide also referred to as "bubblegum" (BLG),
having an amino acid sequence as set forth in SEQ ID NO:2.
[0008] In another embodiment, the present invention provides an
isolated polynucleotide encoding an amino acid sequence as set
forth in SEQ ID NO:2. The isolated polynucleotide is selected from
the group consisting of SEQ ID NO:1; SEQ ID NO:1, wherein T can
also be U; a nucleic acid sequence complementary to SEQ ID NO:1;
and fragments thereof that are at least 15 bases in length and that
hybridize under stringent conditions to DNA which encodes the
polypeptide of SEQ ID NO:2.
[0009] In another embodiment, the present invention provides an
expression vector containing a VLCFA coA-syn polynucleotide. The
vector can be for example, a plasmid or a viral vector.
[0010] In yet another embodiment, the present invention provides a
host cell transformed with an expression vector containing a VLCFA
coA-syn polynucleotide.
[0011] In yet a further embodiment, the present invention provides
a method of producing a VLCFA coA-syn polypeptide by transforming a
host cell with a VLCFA coA-syn polynucleotide; expressing the
polynucleotide in the host; and recovering the VLCFA coA-syn
polypeptide.
[0012] In another embodiment, an antibody that binds to the
polypeptide of SEQ ID NO:2 is provided. The antibody can be
polyclonal or monoclonal.
[0013] The present invention also provides a method for identifying
a compound which modulates VLCFA coA-syn expression or activity
comprising: incubating components comprising the compound and a
VLCFA coA-syn polypeptide, or a recombinant cell expressing a VLCFA
coA-syn polypeptide, under conditions sufficient to allow the
components to interact; and determining the effect of the compound
on the expression or activity of the gene or polypeptide,
respectively.
[0014] In yet another embodiment, the present invention provides a
method of producing a non-human organism having an increased life
span comprising: introducing a transgene disrupting or interfering
with expression of very long chain fatty acid coA-synthetase (VLCFA
coA-syn) into germ cells of a pronuclear embryo of the organism;
implanting the embryo into the oviduct of a pseudopregnant female
thereby allowing the embryo to mature to full term progeny; testing
the progeny for presence of the transgene to identify
transgene-positive progeny; and cross-breeding transgene-positive
progeny to obtain further transgene-positive progeny.
[0015] In yet another embodiment, the present invention provides a
transgenic organisms having a phenotype characterized by
neurodegeneration. The organism may be any non-human organisms,
including, for example, mammals (bovine, porcine) and invertebrates
such as Drosophila.
[0016] These and other aspects of the present invention will be
apparent to those of skill in the art from the teachings
herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The following figures are examples of embodiments and are
not meant to limit the scope of the invention.
[0018] FIG. 1 is a horizontal histological section of the optic
lobe of an adult male fly. (A) Young mutant fly (1 day old). (B) 15
day old mutant. La, Lamina; Re, Retina. Scale bar: 50 .mu.m. (C)
Survival curve of bubblegum males, compared with parental
strain.
[0019] FIG. 2 shows unstructural abnormalities in the bubblegum
mutant. (A) shows the lamina of one day old bubblegum mutant fly.
The photoreceptor axons (arrows), are normal in diameter (indicated
by brackets). (B) In a 15 day old bubblegum, photoreceptor axons
are greatly expanded. (C) tangential section of lamina of one day
old bubblegum mutant shows the normal clustered array of second
order axons surrounded by photoreceptor axons. (D) Fifteen day old
bubblegum at the same magnification, showing the greatly dilated
structure. Inset; tubulovesicular structures within photoreceptor
axons (inset; scale bar: 0.5 .mu.m). L, laminar second order axon;
Pr, photoreceptor axon. Scale bar: 2 .mu.m.
[0020] FIGS. 3A1 and 3A2 show the bubblegum polynucleotide
sequence.
[0021] FIG. 3A3 shows a polynucleotide and polypeptide sequence of
bubblegum VLCFA acyl CoA synthetase. FIGS. 3B1 and 3B2 show an
alignment with rat VLCFA acyl CoA synthetase and a human sequence.
Identical and similar amino acids are identified as boxes.
[0022] FIG. 4 shows the effect of dietary oil-treatment on the
spectrum of very long chain fatty acids in a bubblegum mutant fly.
(A) Without oil treatment. (B) GTO-treated. Each fatty acid was
measured as a percentage of the total. Bars represent ratios of
bubblegum to the parental strain. Both being 15 days old.
[0023] FIG. 5 shows prevention of degeneration and the effect of
glycerol trioleate treatment of bubblegum mutants. (A) bubblegum
mutants fed with GTO for 15 days following emergence. (B) Fifteen
day old adult bubblegum mutants that had been raised in medium
containing GTO from the larval stage. Degeneration largely
prevented. Scale bar: 50 .mu.m. (C) Countercurrent phototaxis
analysis of 5 day old bubblegum. Flies raised without GTO show poor
performance. Flies pretreated with GTO from the larval stage showed
greatly enhanced response to the light.
[0024] FIG. 6 shows a schematic of a pathway identifying the
location and affect the bubblegum mutant has in the degradation of
VLCFAs by .beta.-oxidation. Normally, the synthetase activates
VLCFAs for degradation. In ALD patients a genetic defect in an ABC
transporter interferes with the function of the synthetase. As a
consequence, there is an excess accumulation of VLCFAs. In the
bubblegum mutant, a similar effect is due to a mutation in the
synthetase itself.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention provides polypeptides and
polynucleotides encoding the polypeptides, wherein each polypeptide
is characterized as a neurodegenerative related polypeptide having
very long chain fatty acid coA-synthetase activity.
[0026] The VLCFA CoA-syn has also been termed a bubblegum
polypeptide based upon the appearance of the mutant lamina in
Drosophila. In the lamina (the equivalent in the fly of the
ganglion cell layer in the vertebrate eye), photoreceptor axons
entering from the retina form multiple synapses with a pair of
monopolar, second order neurons. The photoreceptor axons are
readily identified by the presence of capitate projections which,
in sections, resemble golf balls on tees; these project in from
surrounding glial sheaths. As shown in FIG. 2, with age, there is a
great expansion of the photoreceptor axons and other structures,
whereas the capitate projections remain normal in size. At high
magnification, one can see an accumulation of tubulo-vesicular
structures in the expanded axons (FIG. 2d. inset). These resemble
the material that accumulates in vertebrate cerebral white matter
after vinblastine treatment, which causes the destruction of
microtubules (Hirano A., A guide to Neuropathology, Igaku-Shoin,
New York, Tokyo, 1981).
[0027] VLCFA acyl CoA synthetase plays an important role in ALD.
VLCFAs must be activated to their thioester derivatives by the
enzyme before 1-oxidation in peroxisomes. Reduced activity of the
enzyme results in accumulation of VLCFA, especially C26. However,
ALD patients currently have been identified as having mutations not
of the VLCFA acyl CoA enzyme, but in a member of the A BC
transporter superfamily (Mosser et al., Nature, 361:726
(1993)).
[0028] In ALD, dietary treatment has been used in an attempt to
prevent progression of the disease by restoring the normal levels
of VLCFAs. Feeding a monounsaturated fatty acid, glyceryl trioleate
(GTO) oil, reduced the level of C26 in plasm by about 50% within 4
months (Rizzo et al., Neurology, 36:357-361 (1986); Moser et al.,
Ann. Neurol. 21:240-249 (1987)). So called "Lorenzo's oil", a
combination of GTO and glyceryl trierucate, normalized the C26
accumulation in a month. These monounsaturated fatty acids are
thought to compete in the fatty acid elongation system, reducing
the levels of VLCFAs.
[0029] Among ALD patients, the mutations in the ABC transporter are
at various locations within the gene and the clinical phenotype
varies greatly. Even within a single family, the disease can range
from severe to asymptomatic; no relationship has been found between
the expression level of the ABC protein or mutants thereof, and
severity of the disease. In the study by Kok et al., there were two
cases in which the patients were diagnosed clinically as ALD, and
showed high levels of C26, yet had no detectable mutations in the
ABC gene. Burdette et al., diagnosed a patient with neural
disorders, retinal pigmentary degeneration, and high level of C26
as being due to a unique peroxisomal disorder, the clinical
phenotype being between that of ALD and the Zellweger syndrome,
which results from lack of peroxisome function. Three different
knockouts of the gene in mice failed to show demyelination or
paraparesis, although some VLCFAs accumulation was found in the
tissues. It is thought that there may be redundancy of the
ABC-ALD-associated protein in the mice. Alternatively, there may be
one or more other genes involved in determination of onset and
severity of the diseases. Autosomal modifiers that influence the
degree of clinical phenotype in ALD have also been proposed.
[0030] The major biochemical change in the disease is the high
level of accumulation of C26. The .beta.-oxidation of VLCFAs is
catalyzed in several steps by different enzymes in peroxisomes,
VLCFA acyl CoA synthetase, acyl CoA oxidase, bifunctional enoyl-CoA
hydratase/3-hydroxy acyl-CoA dehydratase, and .beta.-ketothilase.
Enzyme disorders in any step of the oxidation processes can result
in elevated levels of VLCFAs, causing peroxisomal diseases. The
mutant bubblegum (described herein) shows the phenotypes associated
with defective VLCFA acyl CoA synthetase, including accumulation of
C26, neurodegeneration, visual impairment, and reduced lifespan,
similar to the effects of mutations in the ABC gene.
[0031] The inventors found that GTO treatment of the mutant flies
prevented the onset of degeneration, concomitant with the VLCFAs
level in males becoming normal. While dietary treatment with
"Lorenzo's oil" in ALD reduces the level of C26, there has been
relatively little success, so far, in preventing progression of the
disease.
[0032] In one embodiment the present invention provides a method
for optimizing the treatment of ALD and associated
neurodegenerative diseases using the mutant bubblegum as a useful
model system for rapidly screening food additives and drugs for
positive effects, and to study the molecular mechanism involved in
neuropathy caused by the unbalance in fatty acid metabolism. At the
same time, the fly could be used to identify suppressor or enhancer
genes that change the phenotype of bubblegum, providing information
on interaction of the synthetase with other genes.
[0033] To facilitate further understanding of the invention, a
number of terms are defined below.
[0034] The term "isolated" means altered "by the hand of man" from
its natural state; i.e., if it occurs in nature, it has been
changed or removed from its original environment, or both. For
example, a naturally occurring polynucleotide or a polypeptide
naturally present in a living animal in its natural state is not
"isolated", but the same polynucleotide or polypeptide separated
from the coexisting materials of its natural state is "isolated",
as the term is employed herein. As part of or following isolation,
a polynucleotide can be joined to other polynucleotides, such as
for example DNAs, for mutagenesis studies, to form fusion proteins,
and for propagation or expression of the polynucleotide in a host.
The isolated polynucleotides, alone or joined to other
polynucleotides, such as vectors, can be introduced into host
cells, in culture or in whole organisms. Such polynucleotides, when
introduced into host cells in culture or in whole organisms, still
would be isolated, as the term is used herein, because they would
not be in their naturally occurring form or environment. Similarly,
the polynucleotides and polypeptides may occur in a composition,
such as a media formulation (solutions for introduction of
polynucleotides or polypeptides, for example, into cells or
compositions or solutions for chemical or enzymatic reactions which
are not naturally occurring compositions) and, therein remain
isolated polynucleotides or polypeptides within the meaning of that
term as it is employed herein.
[0035] The term "oligonucleotide" as used herein is defined as a
molecule comprised of two or more deoxyribonucleotides or
ribonucleotides, preferably more than three, and usually more than
ten. The exact size of an oligonucleotide will depend on many
factors, including the ultimate function or use of the
oligonucleotide. Oligonucleotides can be prepared by any suitable
method, including, for example, cloning and restriction of
appropriate sequences and direct chemical synthesis by a method
such as the phosphotriester method of Narang et al., 1979, Meth.
Enzymol., 68:90-99; the phosphodiester method of Brown et al.,
1979, Method Enzymol., 68:109-151, the diethylphosphoramidite
method of Beaucage et al., 1981, Tetrahedron Lett., 22:1859-1862;
the triester method of Matteucci et al., 1981, J. Am. Chem. Soc.,
103:3185-3191, or automated synthesis methods; and the solid
support method of U.S. Pat. No. 4,458,066.
[0036] The term "plasmids" generally is designated herein by a
lower case p preceded and/or followed by capital letters and/or
numbers, in accordance with standard naming conventions that are
familiar to those of skill in the art. Plasmids disclosed herein
are either commercially available, publicly available on an
unrestricted basis, or can be constructed from available plasmids
by routine application of well known, published procedures. Many
plasmids and other cloning and expression vectors that can be used
in accordance with the present invention are well known and readily
available to those of skill in the art. Moreover, those of skill
readily may construct any number of other plasmids suitable for use
in the invention. The properties, construction and use of such
plasmids, as well as other vectors, in the present invention will
be readily apparent to those of skill from the present
disclosure.
[0037] "Polynucleotide" or "nucleic acid sequence" refers to a
polymeric form of nucleotides at least 10 bases in length. By
"isolated nucleic acid sequence, is meant a polynucleotide that is
not immediately contiguous with either of the coding sequences with
which it is immediately contiguous (one on the 5' end and one on
the 3' end) in the naturally occurring genome of the organism from
which it is derived. The term therefore includes, for example, a
recombinant DNA which is incorporated into a vector; into an
autonomously replicating plasmid or virus; or into the genomic DNA
of a prokaryote or eukaryote, or which exists as a separate
molecule (e.g., a cDNA) independent of other sequences. The
nucleotides of the invention can be ribonucleotides,
deoxyribonucleotides, or modified forms of either nucleotide. The
term includes single and double stranded forms of DNA.
[0038] The term polynucleotide(s) generally refers to any
polyribonucleotide or polydeoxyribonucleotide, which may be
unmodified RNA or DNA or modified RNA or DNA. Thus, for instance,
polynucleotides as used herein refers to, among others, single- and
double-stranded DNA, DNA that is a mixture of single- and
double-stranded regions, single- and double-stranded RNA, and RNA
that is mixture of single- and double-stranded regions, hybrid
molecules comprising DNA and RNA that may be single-stranded or,
more typically, double-stranded or a mixture of single- and
double-stranded regions.
[0039] In addition, polynucleotide as used herein refers to
triple-stranded regions comprising RNA or DNA or both RNA and DNA.
The strands in such regions may be from the same molecule or from
different molecules. The regions may include all of one or more of
the molecules, but more typically involve only a region of some of
the molecules. One of the molecules of a triple-helical region
often is an oligonucleotide.
[0040] As used herein, the term polynucleotide includes DNAs or
RNAs as described above that contain one or more modified bases.
Thus, DNAs or RNAs with backbones modified for stability or for
other reasons are "polynucleotides" as that term is intended
herein. Moreover, DNAs or RNAs comprising unusual bases, such as
inosine, or modified bases, such as tritylated bases, to name just
two examples, are polynucleotides as the term is used herein.
[0041] It will be appreciated that a great variety of modifications
have been made to DNA and RNA that serve many useful purposes known
to those of skill in the art. The term polynucleotide as it is
employed herein embraces such chemically, enzymatically or
metabolically modified forms of polynucleotides, as well as the
chemical forms of DNA and RNA characteristic of viruses and cells,
including simple and complex cells, inter alia.
[0042] Nucleic acid sequences can be created which encode a fusion
protein and can be operatively linked to expression control
sequences. "Operatively linked" refers to a juxtaposition wherein
the components so described are in a relationship permitting them
to function in their intended manner. An expression control
sequence operatively linked to a coding sequence is ligated such
that expression of the coding sequence is achieved under conditions
compatible with the expression control sequences. As used herein,
the term "expression control sequences" refers to nucleic acid
sequences that regulate the expression of a nucleic acid sequence
to which it is operatively linked. Expression control sequences are
operatively linked to a nucleic acid sequence when the expression
control sequences control and regulate the transcription and, as
appropriate, translation of the nucleic acid sequence. Thus,
expression control sequences can include appropriate promoters,
enhancers, transcription terminators, a start codon (i.e., ATG) in
front of a protein-encoding gene, splicing signals for introns,
maintenance of the correct reading frame of that gene to permit
proper translation of the mRNA, and stop codons. The term "control
sequences" is intended to include, at a minimum, components whose
presence can influence expression, and can also include additional
components whose presence is advantageous, for example, leader
sequences and fusion partner sequences. Expression control
sequences can include a promoter.
[0043] By "promoter" is meant minimal sequence sufficient to direct
transcription. Also included in the invention are those promoter
elements which are sufficient to render promoter-dependent gene
expression controllable for cell-type specific, tissue-specific, or
inducible by external signals or agents; such elements may be
located in the 5' or 3' regions of the gene. Both constitutive and
inducible promoters, are included in the invention (see e.g.,
Bitter et al., Methods in Enzymology 153:516-544, 1987). For
example, when cloning in bacterial systems, inducible promoters
such as pL of bacteriophage .gamma., plac, ptrp, ptac (ptrp-lac
hybrid promoter) and the like may be used. When cloning in
mammalian cell systems, promoters derived from the genome of
mammalian cells (e.g., metallothionein promoter) or from mammalian
viruses (e.g., the retrovirus long terminal repeat; the adenovirus
late promoter; the vaccinia virus 7.5K promoter) may be used.
Promoters produced by recombinant DNA or synthetic techniques may
also be used to provide for transcription of the nucleic acid
sequences of the invention.
[0044] A nucleic acid sequence of the invention including, for
example, a polynucleotide encoding a fusion protein, may be
inserted into a recombinant expression vector. The term
"recombinant expression vector" refers to a plasmid, virus or other
vehicle known in the art that has been manipulated by insertion or
incorporation of a nucleic acid sequences of the invention. The
expression vector typically contains an origin of replication, a
promoter, as well as specific genes which allow phenotypic
selection of the transformed cells. Vectors suitable for use in the
present invention include, but are not limited to the T7-based
expression vector for expression in bacteria (Rosenberg, et al.,
Gene 56:125, 1987), the pMSXND expression vector for expression in
mammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988),
baculovirus-derived vectors for expression in insect cells,
cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV. The
nucleic acid sequences of the invention can also include a
localization sequence to direct the indicator to particular
cellular sites by fusion to appropriate organellar targeting
signals or localized host proteins. For example, a polynucleotide
encoding a localization sequence, or signal sequence, can be used
as a repressor and thus can be ligated or fused at the 5' terminus
of a polynucleotide encoding a polypeptide of the invention such
that the localization or signal peptide is located at the amino
terminal end of a resulting polynucleotide/polypeptide. The
construction of expression vectors and the expression of genes in
transfected cells involves the use of molecular cloning techniques
also well known in the art. Sambrook et al., Molecular Cloning--A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y., 1989, and Current Protocols in Molecular Biology, M.
Ausubel et al., eds., (Current Protocols, a joint venture between
Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,
most recent Supplement). These methods include in vitro recombinant
DNA techniques, synthetic techniques and in vivo
recombination/genetic recombination. (See, for example, the
techniques described in Maniatis, et al., Molecular Cloning A
Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1989).
[0045] Depending on the vector utilized, any of a number of
suitable transcription and translation elements, including
constitutive and inducible promoters, transcription enhancer
elements, transcription terminators, etc. may be used in the
expression vector (see, e.g., Bitter, et al., Methods in Enzymology
153:516-544, 1987). These elements are well known to one of skill
in the art.
[0046] In yeast, a number of vectors containing constitutive or
inducible promoters may be used. For a review see, Current
Protocols in Molecular Biology, Vol. 2, Ed. Ausubel, et al., Greene
Publish. Assoc. & Wiley Interscience, Ch. 13, 1988; Grant, et
al., "Expression and Secretion Vectors for Yeast," in Methods in
Enzymology, Eds. Wu & Grossman, 1987, Acad. Press, N.Y., Vol.
153, pp.516-544, 1987; Glover, DNA Cloning, Vol. II, IRL Press,
Wash., D.C., Ch. 3, 1986; and Bitter, "Heterologous Gene Expression
in Yeast," Methods in Enzymology, Eds. Berger & Kimmel, Acad.
Press, N.Y., Vol. 152, pp. 673-684, 1987; and The Molecular Biology
of the Yeast Saccharomyces, Eds. Strathern et al., Cold Spring
Harbor Press, Vols. I and II, 1982. A constitutive yeast promoter
such as ADH or LEU2 or an inducible promoter such as GAL may be
used ("Cloning in Yeast," Ch. 3, R. Rothstein In: DNA Cloning
Vol.11, A Practical Approach, Ed. DM Glover, IRL Press, Wash.,
D.C., 1986). Alternatively, vectors may be used which promote
integration of foreign DNA sequences into the yeast chromosome.
[0047] An alternative expression system which could be used to
express a protein of the invention is an insect system. In one such
system, Autographa californica nuclear polyhedrosis virus (AcNPV)
is used as a vector to express foreign genes. The virus grows in
Spodoptera frugiperda cells. The sequence encoding a protein of the
invention may be cloned into non-essential regions (for example,
the polyhedrin gene) of the virus and placed under control of an
AcNPV promoter (for example the polyhedrin promoter). Successful
insertion of the sequences coding for a protein of the invention
will result in inactivation of the polyhedrin gene and production
of non-occluded recombinant virus (i.e., virus lacking the
proteinaceous coat coded for by the polyhedrin gene). These
recombinant viruses are then used to infect Spodoptera frugiperda
cells in which the inserted gene is expressed, see Smith, et al.,
J. Viol. 46:584, 1983; Smith, U.S. Pat. No. 4,215,051.
[0048] By "transformation" is meant a permanent or transient
genetic change induced in a cell following incorporation of new DNA
(i.e., DNA exogenous to the cell). Where the cell is a mammalian
cell, a permanent genetic change is generally achieved by
introduction of the DNA into the genome of the cell.
[0049] By "transformed cell" or "host cell" is meant a cell (e.g.,
prokaryotic or eukaryotic) into which (or into an ancestor of
which) has been introduced, by means of recombinant DNA techniques,
a DNA molecule encoding a polypeptide of the invention (i.e., a
Very long chain fatty acid coA-synthetase polypeptide), or fragment
thereof.
[0050] Transformation of a host cell with recombinant DNA may be
carried out by conventional techniques as are well known to those
skilled in the art. Where the host is prokaryotic, such as E. coli,
competent cells which are capable of DNA uptake can be prepared
from cells harvested after exponential growth phase and
subsequently treated by the CaCl.sub.2 method by procedures well
known in the art. Alternatively, MgCl.sub.2 or RbCl can be used.
Transformation can also be performed after forming a protoplast of
the host cell or by electroporation.
[0051] When the host is a eukaryote, such methods of transfection
with DNA include calcium phosphate co-precipitates, conventional
mechanical procedures such as microinjection, electroporation,
insertion of a plasmid encased in liposomes, or virus vectors, as
well as others known in the art, may be used. Eukaryotic cells can
also be cotransfected with DNA sequences encoding a polypeptide of
the invention, and a second foreign DNA molecule encoding a
selectable phenotype, such as the herpes simplex thymidine kinase
gene. Another method is to use a eukaryotic viral vector, such as
simian virus 40 (SV40) or bovine papilloma virus, to transiently
infect or transform eukaryotic cells and express the protein.
(Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman
ed., 1982). Preferably, a eukaryotic host is utilized as the host
cell as described herein. The eukaryotic cell may be a yeast cell
(e.g., Saccharomyces cerevisiae), or may be a mammalian cell,
including a human cell.
[0052] Eukaryotic systems, and mammalian expression systems, allow
for proper post-translational modifications of expressed mammalian
proteins to occur. Eukaryotic cells which possess the cellular
machinery for proper processing of the primary transcript,
glycosylation, phosphorylation, and, advantageously secretion of
the gene product should be used. Such host cell lines may include
but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, Jurkat,
HEK-293, and WI38.
[0053] Mammalian cell systems which utilize recombinant viruses or
viral elements to direct expression may be engineered. For example,
when using adenovirus expression vectors, the nucleic acid
sequences encoding a fusion protein of the invention may be ligated
to an adenovirus transcription/translation control complex, e.g.,
the late promoter and tripartite leader sequence. This chimeric
gene may then be inserted in the adenovirus genome by in vitro or
in vivo recombination. Insertion in a non-essential region of the
viral genome (e.g., region E1 or E3) will result in a recombinant
virus that is viable and capable of expressing the Very long chain
fatty acid coA-synthetase polypeptide in infected hosts (e.g., see
Logan & Shenk, Proc. Natl. Acad. Sci. USA, 81:3655-3659, 1984).
Alternatively, the vaccinia virus 7.5K promoter may be used. (e.g.,
see, Mackett, et al., Proc. Natl. Acad. Sci. USA, 79:7415-7419,
1982; Mackett, et al., J. Virol. 49:857-864, 1984; Panicali, et
al., Proc. Natl. Acad. Sci. USA 79:4927-4931, 1982). Of particular
interest are vectors based on bovine papilloma virus which have the
ability to replicate as extrachromosomal elements (Sarver, et al.,
Mol. Cell. Biol. 1:486, 1981). Shortly after entry of this DNA into
mouse cells, the plasmid replicates to about 100 to 200 copies per
cell. Transcription of the inserted cDNA does not require
integration of the plasmid into the host's chromosome, thereby
yielding a high level of expression. These vectors can be used for
stable expression by including a selectable marker in the plasmid,
such as the neo gene. Alternatively, the retroviral genome can be
modified for use as a vector capable of introducing and directing
the expression of the Very long chain fatty acid coA-synthetase
gene in host cells (Cone & Mulligan, Proc. Natl. Acad. Sci.
USA, 81:6349-6353, 1984). High level expression may also be
achieved using inducible promoters, including, but not limited to,
the metallothionine IIA promoter and heat shock promoters.
[0054] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. Rather than using
expression vectors which contain viral origins of replication, host
cells can be transformed with the cDNA encoding a fusion protein of
the invention controlled by appropriate expression control elements
(e.g., promoter, enhancer, sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. The
selectable marker in the recombinant plasmid confers resistance to
the selection and allows cells to stably integrate the plasmid into
their chromosomes and grow to form foci which in turn can be cloned
and expanded into cell lines.
[0055] For example, following the introduction of foreign DNA,
engineered cells may be allowed to grow for 1-2 days in an enriched
media, and then are switched to a selective media. A number of
selection systems may be used, including but not limited to the
herpes simplex virus thymidine kinase (Wigler, et al., Cell,
11:223, 1977), hypoxanthine-guanine phosphoribosyltransferase
(Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48:2026,
1962), and adenine phosphoribosyltransferase (Lowy, et al., Cell,
22:817, 1980) genes can be employed in tk.sup.-, hgprt.sup.- or
aprt.sup.- cells respectively. Also, antimetabolite resistance can
be used as the basis of selection for dhfr, which confers
resistance to methotrexate (Wigler, et al., Proc. Natl. Acad. Sci.
USA, 77:3567, 1980; O'Hare, et al., Proc. Natl. Acad. Sci. USA,
8:1527, 1981); gpt, which confers resistance to mycophenolic acid
(Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072, 1981;
neo, which confers resistance to the aminoglycoside G-418
(Colberre-Garapin, et al., J. Mol. Biol. 150:1, 1981); and hygro,
which confers resistance to hygromycin (Santerre, et al., Gene
30:147, 1984) genes. Recently, additional selectable genes have
been described, namely trpB, which allows cells to utilize indole
in place of tryptophan; hisD, which allows cells to utilize
histinol in place of histidine (Hartman & Mulligan, Proc. Natl.
Acad. Sci. USA 85:8047, 1988); and ODC (ornithine decarboxylase)
which confers resistance to the ornithine decarboxylase inhibitor,
2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L., In: Current
Communications in Molecular Biology, Cold Spring Harbor Laboratory,
ed., 1987).
[0056] The term "primer" as used herein refers to an
oligonucleotide, whether natural or synthetic, which is capable of
acting as a point of initiation of synthesis when placed under
conditions in which primer extension is initiated or possible.
Synthesis of a primer extension product which is complementary to a
nucleic acid strand is initiated in the presence of nucleoside
triphosphates and a polymerase in an appropriate buffer at a
suitable temperature. The term "primer" may refer to more than one
primer, particularly in the case where there is some ambiguity in
the information regarding one or both ends of the target region to
be synthesized. For instance, if a nucleic acid sequence is
inferred from a protein sequence, a "primer" generated to
synthesize nucleic acid sequence encoding the protein sequence is
actually a collection of primer oligonucleotides containing
sequences representing all possible codon variations based on the
degeneracy of the genetic code. One or more of the primers in this
collection will be homologous with the end of the target sequence.
Likewise, if a "conserved" region shows significant levels of
polymorphism in a population, mixtures of primers can be prepared
that will amplify adjacent sequences. For example, primers can be
synthesized based upon the amino acid sequence as set forth in SEQ
ID NO:2 and can be designed based upon the degeneracy of the
genetic code.
[0057] The term "gene" means the segment of DNA involved in
producing a polypeptide chain; it includes regions preceding and
following the coding region (leader and trailer) as well as
intervening sequences (introns) between individual coding segments
(exons).
[0058] A coding sequence is "operably linked" to another coding
sequence when RNA polymerase will transcribe the two coding
sequences into a single mRNA, which is then translated into a
single polypeptide having amino acids derived from both coding
sequences. The coding sequences need not be contiguous to one
another so long as the expressed sequences ultimately process to
produce the desired protein.
[0059] A "recombinant" protein or polypeptide refer to proteins or
polypeptides produced by recombinant DNA techniques; i.e., produced
from cells transformed by an exogenous DNA construct encoding the
desired polypeptide (e.g. a Very long chain fatty acid
coA-synthetase polypeptide of the present invention). "Synthetic"
polypeptides are those prepared by chemical synthesis.
[0060] As used in connection with the present invention the term
"polypeptide" or "protein" refers to a polymer in which the
monomers are amino acid residues which are joined together through
amide bonds. When the amino acids are alpha-amino acids, either the
L-optical isomer or the D-optical isomer can be used, the L-isomers
being preferred. The term "polypeptide" as used herein is intended
to encompass any amino acid sequence and include modified sequences
such as glycoproteins. The term "polypeptide" is specifically
intended to cover naturally occurring proteins, as well as those
which are recombinantly or synthetically synthesized, which occur
in at least two different conformations wherein both conformations
have the same or substantially the same amino acid sequence but
have different three dimensional structures. "Fragments" are a
portion of a naturally occurring protein. Fragments can have the
same or substantially the same amino acid sequence as the naturally
occurring protein. "Substantially the same" means that an amino
acid sequence is largely, but not entirely, the same, but retains a
functional activity of the sequence to which it is related. In
general, two amino acid sequences are "substantially the same" or
"substantially homologous" if they are at least 70% identical. The
term "conservative variation" as used herein denotes the
replacement of an amino acid residue by another, biologically
similar residue. Examples of conservative variations include the
substitution of one hydrophobic residue such as isoleucine, valine,
leucine or methionine for another, or the substitution of one polar
residue for another, such as the substitution of arginine for
lysine, glutamic for aspartic acids, or glutamine for asparagine,
and the like. Other illustrative examples of conservative
substitutions include the changes of: alanine to serine; arginine
to lysine; asparagine to glutamine or histidine; aspartate to
glutamate; cysteine to serine; glutamine to asparagine; glutamate
to aspartate; glycine to proline; histidine to asparagine or
glutamine; isoleucine to leucine or valine; leucine to valine or
isoleucine; lysine to arginine, glutamine, or glutamate; methionine
to leucine or isoleucine; phenylalanine to tyrosine, leucine or
methionine; serine to threonine; threonine to serine; tryptophan to
tyrosine; tyrosine to tryptophan or phenylalanine; valine to
isoleucine to leucine. The term "conservative variation" also
includes the use of a substituted amino acid in place of an
unsubstituted parent amino acid provided that antibodies raised to
the substituted polypeptide also immunoreact with the unsubstituted
polypeptide.
[0061] Modifications and substitutions are not limited to
replacement of amino acids. For a variety of purposes, such as
increased stability, solubility, or configuration concerns, one
skilled in the art will recognize the need to introduce, (by
deletion, replacement, or addition) other modifications. Examples
of such other modifications include incorporation of rare amino
acids, dextra-amino acids, glycosylation sites, cytosine for
specific disulfide bridge formation, for example of possible
modifications. The modified peptides can be chemically synthesized,
or the isolated gene can be site-directed mutagenized, or a
synthetic gene can be synthesized and expressed in bacteria, yeast,
baculovirus, tissue culture and so on.
[0062] A DNA "coding sequence of" or a "nucleotide sequence
encoding" a particular protein, is a DNA sequence which is
transcribed and translated into an protein when placed under the
control of appropriate regulatory sequences.
[0063] VLCFA coA-Synthetase Nucleic Acid, Polypeptides and Method
of Expression
[0064] In one embodiment, the invention provides an isolated
polynucleotide sequence encoding a VLCFA coA-syn polypeptide. An
exemplary VLCFA coA-syn polypeptide of the invention has an amino
acid sequence as set forth in SEQ ID NO:2. Polynucleotide sequences
of the invention include DNA, cDNA and RNA sequences which encode
VLCFA coA-syn. It is understood that all polynucleotides encoding
all or a portion of VLCFA coA-syn are also included herein, so long
as they encode a polypeptide with VLCFA coA-syn activity (e.g.,
increased life span or resistance to stress). Such polynucleotides
include naturally occurring, synthetic, and intentionally
manipulated polynucleotides. For example, VLCFA coA-syn
polynucleotide may be subjected to site-directed mutagenesis. The
polynucleotides of the invention include sequences that are
degenerate as a result of the genetic code. There are 20 natural
amino acids, most of which are specified by more than one codon.
Therefore, all degenerate nucleotide sequences are included in the
invention so long as the amino acid sequence of VLCFA coA-syn
polypeptide encoded by the nucleotide sequence is functionally
unchanged. Also included are nucleotide sequences which encode
VLCFA coA-syn polypeptide, such as SEQ ID NO:1. In addition, the
invention also includes a polynucleotide encoding a polypeptide
having the biological activity of an amino acid sequence of SEQ ID
NO:2 and having at least one epitope for an antibody immunoreactive
with VLCFA coA-syn polypeptide. However, it is recognized that
portions of either SEQ ID NO:1 or 2 may be excluded to identify
fragments of the polynucleotide sequence or polypeptide sequence.
For example, fragments of SEQ ID NO:1 or 2 are encompassed by the
current invention, so long as they retain some biological activity
related to VLCFA coA-syn. A biological activity related to VLCFA
coA-syn includes for example, antigenicity or the ability to
regulate .beta.-oxidation of fatty acids.
[0065] The polynucleotides of this invention were originally
recovered from Drosophila melanogaster. Thus, the present invention
provides means for isolating the nucleic acid molecules from other
organisms, including humans, encoding the polypeptides of the
present invention. For example, one may probe a gene library with a
natural or artificially designed probe using art recognized
procedures (see, for example: Current Protocols in Molecular
Biology, Ausubel F. M. et al. (EDS.) Green Publishing Company
Assoc. and John Wiley Interscience, New York, 1989, 1992). It is
appreciated by one skilled in the art that probes can be designed
based on the degeneracy of the genetic code to the sequences set
forth in SEQ ID NO:2.
[0066] The invention includes polypeptides having substantially the
same sequence as the amino acid sequence set forth in SEQ ID NO:2
or functional fragments thereof, or amino acid sequences that are
substantially identical or the same as SEQ ID NO:2.
[0067] Homology or identity is often measured using sequence
analysis software (e.g., Sequence Analysis Software Package of the
Genetics Computer Group, University of Wisconsin Biotechnology
Center, 1710 University Avenue, Madison, Wis. 53705). Such software
matches similar sequences by assigning degrees of homology to
various deletions, substitutions and other modifications. The terms
"homology" and "identity" in the context of two or more nucleic
acids or polypeptide sequences, refer to two or more sequences or
subsequences that are the same or have a specified percentage of
amino acid residues or nucleotides that are the same when compared
and aligned for maximum correspondence over a comparison window or
designated region as measured using any number of sequence
comparison algorithms or by manual alignment and visual
inspection.
[0068] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0069] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequence for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol 48:443 (1970), by
the search for similarity method of person & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection.
[0070] One example of a useful algorithm is BLAST and BLAST 2.0
algorithms, which are described in Altschul et al., Nuc. Acids Res.
25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410
(1990), respectively. Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information on the world wide web at ncbi.nlm.nih.gov. This
algorithm involves first identifying high scoring sequence pairs
(HSPs) by identifying short words of length W in the query
sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul et al., supra). These initial neighborhood word
hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) or 11, an expectation
(E) or 10, M=5, N=-4 and a comparison of both strands. For amino
acid sequences, the BLASTP program uses as defaults a wordlength of
3, and expectations (E) of 10, and the BLOSUM62 scoring matrix (see
Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915
(1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and
a comparison of both strands.
[0071] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin &
Altschul, Proc. Natl. Acad. Sci. USA 90:5873 (1993)). One measure
of similarity provided by BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a references sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.2, more preferably less than about 0.01, and
most preferably less than about 0.001.
[0072] A "substantially pure polypeptide" is typically pure when it
is at least 60%, by weight, free from the proteins and
naturally-occurring organic molecules with which it is naturally
associated. Preferably, the preparation is at least 75%, more
preferably at least 90%, and most preferably at least 99%, by
weight, VLCFA coA-syn polypeptide. A substantially pure VLCFA
coA-syn polypeptide may be obtained, for example, by extraction
from a natural source (e.g., an insect cell); by expression of a
recombinant nucleic acid encoding an VLCFA coA-syn polypeptide; or
by chemically synthesizing the protein. Purity can be measured by
any appropriate method, e.g., by column chromatography,
polyacrylamide gel electrophoresis, or by HPLC analysis.
[0073] VLCFA coA-syn polypeptides of the present invention include
peptides, or full length protein, that contains substitutions,
deletions, or insertions into the protein backbone, that would
still leave an approximately 50%-70% homology to the original
protein over the corresponding portion. A yet greater degree of
departure from homology is allowed if like-amino acids, i.e.
conservative amino acid substitutions, do not count as a change in
the sequence.
[0074] In addition to polypeptides of the invention, specifically
disclosed herein is a DNA sequence for VLCFA coA-syn represented by
SEQ ID NO:1. DNA sequences of the invention can be obtained by
several methods. For example, the DNA can be isolated using
hybridization or computer-based techniques which are well known in
the art. These include, but are not limited to: 1) hybridization of
genomic libraries with probes to detect homologous nucleotide
sequences; 2) antibody screening of expression libraries to detect
cloned DNA fragments with shared structural features; 3) polymerase
chain reaction (PCR) on genomic DNA using primers capable of
annealing to the DNA sequence of interest; and 4) computer searches
of sequence databases for similar sequences.
[0075] The polynucleotide encoding VLCFA coA-syn includes the
nucleotide sequence in FIG. 3 (SEQ ID NO:1), as well as nucleic
acid sequences complementary to that sequence. When the sequence is
RNA, the deoxyribonucleotides A, G, C, and T of SEQ ID NO:1 are
replaced by ribonucleotides A, G, C, and U, respectively. Also
included in the invention are fragments (portions) of the
above-described nucleic acid sequences that are at least 15 bases
in length, which is sufficient to permit the fragment to
selectively hybridize to DNA that encodes the protein of FIG. 3
(e.g., SEQ ID NO: 2). "Selective hybridization" as used herein
refers to hybridization under moderately stringent or highly
stringent physiological conditions (See, for example, the
techniques described in Maniatis et al., 1989 Molecular Cloning A
Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.,
incorporated herein by reference), which distinguishes related from
unrelated nucleotide sequences.
[0076] In nucleic acid hybridization reactions, the conditions used
to achieve a particular level of stringency will vary, depending on
the nature of the nucleic acids being hybridized. For example, the
length, degree of complementarity, nucleotide sequence composition
(e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA)
of the hybridizing regions of the nucleic acids can be considered
in selecting hybridization conditions. An additional consideration
is whether one of the nucleic acids is immobilized, for example, on
a filter.
[0077] An example of progressively higher stringency conditions is
as follows: 2.times.SSC/0.1% SDS at about room temperature
(hybridization conditions); 0.2.times.SSC/0.1% SDS at about room
temperature (low stringency conditions); 0.2.times.SSC/0.1% SDS at
about 42.degree. C. (moderate stringency conditions); and
0.1.times.SSC at about 68.degree. C. (high stringency conditions).
Washing can be carried out using only one of these conditions,
e.g., high stringency conditions, or each of the conditions can be
used, e.g., for 10-15 minutes each, in the order listed above,
repeating any or all of the steps listed. However, as mentioned
above, optimal conditions will vary, depending on the particular
hybridization reaction involved, and can be determined
empirically.
[0078] Oligonucleotides encompassed by the present invention are
also useful as primers for nucleic acid amplification reactions. In
general, the primers used according to the method of the invention
embrace oligonucleotides of sufficient length and appropriate
sequence which provides specific initiation of polymerization of a
significant number of nucleic acid molecules containing the target
nucleic acid under the conditions of stringency for the reaction
utilizing the primers. In this manner, it is possible to
selectively amplify the specific target nucleic acid sequence
containing the nucleic acid of interest. Specifically, the term
"primer" as used herein refers to a sequence comprising two or more
deoxyribonucleotides or ribonucleotides, preferably at least eight,
which sequence is capable of initiating synthesis of a primer
extension product that is substantially complementary to a target
nucleic acid strand. The oligonucleotide primer typically contains
15-22 or more nucleotides, although it may contain fewer
nucleotides as long as the primer is of sufficient specificity to
allow essentially only the amplification of the specifically
desired target nucleotide sequence (i.e., the primer is
substantially complementary).
[0079] Amplified products may be detected by Southern blot
analysis, without using radioactive probes. In such a process, for
example, a small sample of DNA containing a very low level of VLCFA
coA-syn nucleotide sequence is amplified and analyzed via a
Southern blotting technique known to those of skill in the art. The
use of non-radioactive probes or labels is facilitated by the high
level of the amplified signal.
[0080] VLCFA coA-syn polynucleotide of the invention is derived
from an insect (e.g., Drosophila). Screening procedures which rely
on nucleic acid hybridization make it possible to isolate any gene
sequence from any organism, provided the appropriate probe is
available. For example, it is envisioned that such probes can be
used to identify other homologs of the VLCFA coA-syn family of
factors in insects or, alternatively, in other organisms such as
mammals, e.g., humans. In accomplishing this, oligonucleotide
probes, which correspond to a part of the sequence encoding the
protein in question, can be synthesized chemically. This requires
that short, oligopeptide stretches of amino acid sequence must be
known. The DNA sequence encoding the protein can be deduced from
the genetic code, however, the degeneracy of the code must be taken
into account. It is possible to perform a mixed addition reaction
when the sequence is degenerate. This includes a heterogeneous
mixture of denatured double-stranded DNA. For such screening,
hybridization is preferably performed on either single-stranded DNA
or denatured double-stranded DNA. Hybridization is particularly
useful in the detection of DNA clones derived from sources where an
extremely low amount of mRNA sequences relating to the polypeptide
of interest are present. In other words, by using stringent
hybridization conditions directed to avoid non-specific binding, it
is possible, for example, to allow the autoradiographic
visualization of a specific cDNA clone by the hybridization of the
target DNA to that single probe in the mixture which is its
complete complement (Wallace, et al., Nucl. Acid Res., 9:879,
1981).
[0081] When the entire sequence of amino acid residues of the
desired polypeptide is not known, the direct synthesis of DNA
sequences is not possible and the method of choice is use of cDNA
sequences. Among the standard procedures for isolating cDNA
sequences of interest is the formation of plasmid- or
phage-carrying cDNA libraries which are derived from reverse
transcription of mRNA which is abundant in donor cells that have a
high level of genetic expression. When used in combination with
polymerase chain reaction technology, even rare expression products
can be cloned.
[0082] DNA sequences encoding VLCFA coA-syn can be expressed in
vitro by DNA transfer into a suitable host cell. "Host cells" are
cells in which a vector can be propagated and its DNA expressed.
The term also includes any progeny of the subject host cell. It is
understood that all progeny may not be identical to the parental
cell since there may be mutations that occur during replication.
However, such progeny are included when the term "host cell" is
used.
[0083] In the present invention, the VLCFA coA-syn polynucleotide
sequences may be inserted into a recombinant expression vector. The
term "recombinant expression vector" refers to a plasmid, virus or
other vehicle known in the art that has been manipulated by
insertion or incorporation of the VLCFA coA-syn genetic sequences.
Such expression vectors contain a promoter sequence which
facilitates the efficient transcription of the inserted genetic
sequence of the host. The expression vector typically contains an
origin of replication, a promoter, as well as specific genes which
allow phenotypic selection of the transformed cells. Vectors
suitable for use in the present invention include those described
above.
[0084] Polynucleotide sequences encoding VLCFA coA-syn can be
expressed in either prokaryotes or eukaryotes. Hosts can include
microbial, yeast, insect and mammalian organisms. Such vectors are
used to incorporate DNA sequences of the invention.
[0085] Methods which are well known to those skilled in the art can
be used to construct expression vectors containing the VLCFA
coA-syn coding sequence and appropriate
transcriptional/translational control signals. These methods
include in vitro recombinant DNA techniques, synthetic techniques,
and in vivo recombination/genetic techniques. (See, for example,
the techniques described in Maniatis et al., 1989, Molecular
Cloning A Laboratory Manual, Cold Spring Harbor Laboratory,
N.Y.)
[0086] The genetic construct can be designed to provide additional
benefits, such as, for example addition of C-terminal or N-terminal
amino acid residues that would facilitate purification by trapping
on columns or by use of antibodies. All those methodologies are
cumulative. For example, a synthetic gene can later be mutagenized.
The choice as to the method of producing a particular construct can
easily be made by one skilled in the art based on practical
considerations: size of the desired peptide, availability and cost
of starting materials, etc. All the technologies involved are well
established and well known in the art. See, for example, Ausubel et
al., Current Protocols in Molecular Biology, Volumes 1 and 2
(1987), with supplements, and Maniatis et al., Molecular Cloning, a
Laboratory Manual, Cold Spring Harbor Laboratory (1989). Yet other
technical references are known and easily accessible to one skilled
in the art.
[0087] Antibodies that Bind to VLCFA coA-syn
[0088] In another embodiment, the present invention provides
antibodies that bind to VLCFA coA-syn. Such antibodies are useful
for research and diagnostic tools in the study of
neurodegeneration, ALD and VLCFA coA-synthetase associated
pathologies in general. Such antibodies may be administered alone
or contained in a pharmaceutical composition comprising antibodies
against VLCFA coA-syn and other reagents effective as modulators
neurodegeneration, 1-oxidation of fatty acids both in vitro and in
vivo.
[0089] The term "epitope", as used herein, refers to an antigenic
determinant on an antigen, such as a VLCFA coA-syn polypeptide, to
which the paratope of an antibody, such as an VLCFA
coA-syn-specific antibody, binds. Antigenic determinants usually
consist of chemically active surface groupings of molecules, such
as amino acids or sugar side chains, and can have specific
three-dimensional structural characteristics, as well as specific
charge characteristics.
[0090] Antibodies which bind to the VLCFA coA-syn polypeptide of
the invention can be prepared using an intact polypeptide or
fragments containing small peptides of interest as the immunizing
antigen. The polypeptide or a peptide used to immunize an animal
can be derived from translated cDNA or chemical synthesis which can
be conjugated to a carrier protein, if desired. Such commonly used
carriers which are chemically coupled to the peptide include
keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum
albumin (BSA), and tetanus toxoid. The coupled peptide is then used
to immunize the animal (e.g., a mouse, a rat, or a rabbit).
[0091] If desired, polyclonal or monoclonal antibodies can be
further purified, for example, by binding to and elution from a
matrix to which the polypeptide or a peptide to which the
antibodies were raised is bound. Those of skill in the art will
know of various techniques common in the immunology arts for
purification and/or concentration of polyclonal antibodies, as well
as monoclonal antibodies (See for example, Coligan, et al., Unit 9,
Current Protocols in Immunology, Wiley Interscience, 1991,
incorporated by reference).
[0092] It is also possible to use the anti-idiotype technology to
produce monoclonal antibodies which mimic an epitope. For example,
an anti-idiotypic monoclonal antibody made to a first monoclonal
antibody will have a binding domain in the hypervariable region
which is the "image" of the epitope bound by the first monoclonal
antibody.
[0093] An antibody suitable for binding to VLCFA coA-syn is
specific for at least one portion of a region of the VLCFA coA-syn
polypeptide, as shown in FIG. 3 (SEQ ID NO:2). For example, one of
skill in the art can use the peptides to generate appropriate
antibodies of the invention. Antibodies of the invention include
polyclonal antibodies, monoclonal antibodies, and fragments of
polyclonal and monoclonal antibodies.
[0094] The preparation of polyclonal antibodies is well-known to
those skilled in the art. See, for example, Green et al.,
Production of Polyclonal Antisera, in Immunochemical Protocols
(Manson, ed.), pages 1-5 (Humana Press 1992); Coligan et al.,
Production of Polyclonal Antisera in Rabbits, Rats, Mice and
Hamsters, in Current Protocols in Immunology, section 2.4.1 (1992),
which are hereby incorporated by reference.
[0095] The preparation of monoclonal antibodies likewise is
conventional. See, for example, Kohler & Milstein, Nature,
256:495 (1975); Coligan et al., sections 2.5.1-2.6.7; and Harlow et
al., Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor
Pub. 1988), which are hereby incorporated by reference. Briefly,
monoclonal antibodies can be obtained by injecting mice with a
composition comprising an antigen, verifying the presence of
antibody production by removing a serum sample, removing the spleen
to obtain B lymphocytes, fusing the B lymphocytes with myeloma
cells to produce hybridomas, cloning the hybridomas, selecting
positive clones that produce antibodies to the antigen, and
isolating the antibodies from the hybridoma cultures. Monoclonal
antibodies can be isolated and purified from hybridoma cultures by
a variety of well-established techniques. Such isolation techniques
include affinity chromatography with Protein-A Sepharose,
size-exclusion chromatography, and ion-exchange chromatography.
See, e.g., Coligan et al., sections 2.7.1-2.7.12 and sections
2.9.1-2.9.3; Barnes et al., Purification of Immunoglobulin G (IgG),
in Methods in Molecular Biology, Vol. 10, pages 79-104 (Humana
Press 1992). Methods of in vitro and in vivo multiplication of
monoclonal antibodies is well-known to those skilled in the art.
Multiplication in vitro may be carried out in suitable culture
media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium,
optionally replenished by a mammalian serum such as fetal calf
serum or trace elements and growth-sustaining supplements such as
normal mouse peritoneal exudate cells, spleen cells, bone marrow
macrophages. Production in vitro provides relatively pure antibody
preparations and allows scale-up to yield large amounts of the
desired antibodies. Large scale hybridoma cultivation can be
carried out by homogenous suspension culture in an airlift reactor,
in a continuous stirrer reactor, or in immobilized or entrapped
cell culture. Multiplication in vivo may be carried out by
injecting cell clones into mammals histocompatible with the parent
cells, e.g., osyngeneic mice, to cause growth of antibody-producing
tumors. Optionally, the animals are primed with a hydrocarbon,
especially oils such as pristane (tetramethylpentadecane) prior to
injection. After one to three weeks, the desired monoclonal
antibody is recovered from the body fluid of the animal.
[0096] Therapeutic applications for antibodies disclosed herein are
also part of the present invention. For example, antibodies of the
present invention may also be derived from subhuman primate
antibody. General techniques for raising therapeutically useful
antibodies in baboons can be found, for example, in Goldenberg et
al., International Patent Publication WO 91/11465 (1991) and Losman
et al., Int. J. Cancer, 46:310 (1990), which are hereby
incorporated by reference.
[0097] Alternatively, an; anti-VLCFA coA-syn antibody may be
derived from a "humanized" monoclonal antibody. Humanized
monoclonal antibodies are produced by transferring mouse
complementarity determining regions from heavy and light variable
chains of the mouse immunoglobulin into a human variable domain,
and then substituting human residues in the framework regions of
the murine counterparts. The use of antibody components derived
from humanized monoclonal antibodies obviates potential problems
associated with the immunogenicity of murine constant regions.
General techniques for cloning murine immunoglobulin variable
domains are described, for example, by Orlandi et al., Proc. Nat'l
Acad. Sci. USA, 86:3833 (1989), which is hereby incorporated in its
entirety by reference. Techniques for producing humanized
monoclonal antibodies are described, for example, by Jones et al.,
Nature, 321: 522 (1986); Riechmann et al., Nature, 332: 323 (1988);
Verhoeyen et al., Science, 239:1534 (1988); Carter et al., Proc.
Nat'l Acad. Sci. USA, 89:4285 (1992); Sandhu, Crit. Rev. Biotech.,
12:437 (1992); and Singer et al., J. Immunol., 150:2844 (1993),
which are hereby incorporated by reference.
[0098] Antibodies of the invention also may be derived from human
antibody fragments isolated from a combinatorial immunoglobulin
library. See, for example, Barbas et al., Methods: A Companion to
Methods in Enzymology, Vol. 2, page 119 (1991); Winter et al., Ann.
Rev. Immunol. 12: 433 (1994), which are hereby incorporated by
reference. Cloning and expression vectors that are useful for
producing a human immunoglobulin phage library can be obtained, for
example, from STRATAGENE Cloning Systems (La Jolla, Calif.).
[0099] In addition, antibodies of the present invention may be
derived from a human monoclonal antibody. Such antibodies are
obtained from transgenic mice that have been "engineered" to
produce specific human antibodies in response to antigenic
challenge. In this technique, elements of the human heavy and light
chain loci are introduced into strains of mice derived from
embryonic stem cell lines that contain targeted disruptions of the
endogenous heavy and light chain loci. The transgenic mice can
synthesize human antibodies specific for human antigens, and the
mice can be used to produce human antibody-secreting hybridomas.
Methods for obtaining human antibodies from transgenic mice are
described by Green et al., Nature Genet., 7:13 (1994); Lonberg et
al., Nature, 368:856 (1994); and Taylor et al., Int. Immunol.,
6:579 (1994), which are hereby incorporated by reference.
[0100] Antibody fragments of the present invention can be prepared
by proteolytic hydrolysis of the antibody or by expression in E.
coli of DNA encoding the fragment. Antibody fragments can be
obtained by pepsin or papain digestion of whole antibodies by
conventional methods. For example, antibody fragments can be
produced by enzymatic cleavage of antibodies with pepsin to provide
a 5S fragment denoted F(ab').sub.2. This fragment can be further
cleaved using a thiol reducing agent, and optionally a blocking
group for the sulfhydryl groups resulting from cleavage of
disulfide linkages, to produce 3.5S Fab' monovalent fragments.
Alternatively, an enzymatic cleavage using pepsin produces two
monovalent Fab' fragments and an Fc fragment directly. These
methods are described, for example, by Goldenberg, U.S. Pat. No.
4,036,945 and No. 4,331,647, and references contained therein.
These patents are hereby incorporated in their entireties by
reference. See also Nisonhoff et al., Arch. Biochem. Biophys,.
89:230 (1960); Porter, Biochem. J., 73:119 (1959); Edelman et al.,
Methods in Enzymology, Vol. 1, page 422 (Academic Press 1967); and
Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4.
[0101] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical, or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0102] For example, Fv fragments comprise an association of V.sub.H
and V.sub.L chains. This association may be noncovalent, as
described in Inbar et al., Proc. Nat'l Acad. Sci. USA, 69:2659
(1972). Alternatively, the variable chains can be linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
glutaraldehyde. See, e.g., Sandhu, supra. Preferably, the Fv
fragments comprise V.sub.H and V.sub.L chains connected by a
peptide linker. These single-chain antigen binding proteins (sFv)
are prepared by constructing a structural gene comprising DNA
sequences encoding the V.sub.H and V.sub.L domains connected by an
oligonucleotide. The structural gene is inserted into an expression
vector, which is subsequently introduced into a host cell such as
E. coli. The recombinant host cells synthesize a single polypeptide
chain with a linker peptide bridging the two V domains. Methods for
producing sFvs are described, for example, by Whitlow et al.,
Methods: A Companion to Methods in Enzymology, Vol. 2, page 97
(1991); Bird et al., Science, 242:423 (1988); Ladner et al., U.S.
Pat. No. 4,946,778; Pack et al., Bio/Technology, 11:1271 (1993);
and Sandhu, supra.
[0103] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing
cells. See, for example, Larrick et al., Methods: A Companion to
Methods in Enzymology, Vol. 2, page 106 (1991).
[0104] Modulation of Neurodegeneration or Survival
[0105] In one embodiment, the invention provides a method for
modulating (e.g., reducing) neurodegeneration in a cell or a
subject by administering to the cell or subject a therapeutically
effective amount of a composition which contains an VLCFA coA-syn
polypeptide, or biologically functional fragment thereof or an
agent (e.g, an antibody, ribozyme, antisense molecule, or
double-stranded interferring RNA molecules). The term "biologically
functional fragment" encompasses any segment of a VLCFA coA-syn
polypeptide that retains the ability to modulate (e.g., increase or
decrease) .beta.-oxidation of VLCFA or which retains acyl-coA
sythetase activity.
[0106] As used herein, a "therapeutically effective amount" of a
composition containing VLCFA coA-syn or an VLCFA coA-syn-modulating
agent is defined as that amount that is effective in modulating
.beta.-oxidation of VLCFAs.
[0107] In another embodiment, the present invention provides a
method for modulating VLCFA coA-syn gene expression and well as
methods for screening for agents which modulate VLCFA coA-syn gene
expression. A cell or subject is contacted with an agent suspected
or known to have VLCFA coA-syn gene expression modulating activity.
The change in VLCFA coA-syn gene expression is then measured as
compared to a control or standard sample. The control or standard
sample can be the baseline expression of the cell or subject prior
to contact with the agent. An agent which modulates VLCFA coA-syn
gene expression may be a polynucleotide for example. The
polynucleotide may be an antisense, a triplex agent, a ribozyme, or
a double-stranded interferring RNA. For example, an antisense may
be directed to the structural gene region or to the promoter region
of VLCFA coA-syn. The agent may be an agonist, antagonist, peptide,
peptidomimetic, antibody, or chemical.
[0108] Double-stranded interferring RNA molecules are especially
useful in the present invention. Such molecules act to inhibit
expression of a target gene. For example, double-stranded RNA
molecules can be injected into a target cell or organism to inhibit
expression of a gene and the resultant gene products activity. It
has been found that such double-stranded RNA molecules are more
effective at inhibiting expression than either RNA strand alone.
(Fire et al., Nature, 1998, 19:391(6669):806-11).
[0109] When a disorder is associated with abnormal expression of
VLCFA coA-syn, a therapeutic approach which directly interferes
with the translation of VLCFA coA-syn messages into protein is
possible. Alternatively, similar methodology may be used to study
VLCFA coA-syn gene activity. For example, antisense nucleic acid,
double-stranded interferring RNA or ribozymes could be used to bind
to the VLCFA coA-syn mRNA or to cleave it. Antisense RNA or DNA
molecules bind specifically with a targeted gene's RNA message,
interrupting the expression of that gene's protein product. The
antisense binds to the messenger RNA forming a double stranded
molecule which cannot be translated by the cell. Antisense
oligonucleotides of about 15-25 nucleotides are preferred since
they are easily synthesized and have an inhibitory effect just like
antisense RNA molecules. In addition, chemically reactive groups,
such as iron-linked ethylenediaminetetraacetic acid (EDTA-Fe) can
be attached to an antisense oligonucleotide, causing cleavage of
the RNA at the site of hybridization. These and other uses of
antisense methods to inhibit the in vitro translation of genes are
well known in the art (Marcus-Sakura, Anal. Biochem., 172:289,
1988).
[0110] Antisense nucleic acids are DNA or RNA molecules that are
complementary to at least a portion of a specific mRNA molecule
(Weintraub, Scientific American, 262:40, 1990). In the cell, the
antisense nucleic acids hybridize to the corresponding mRNA,
forming a double-stranded molecule. The antisense nucleic acids
interfere with the translation of the mRNA, since the cell will not
translate a mRNA that is double-stranded. Antisense oligomers of
about 15 nucleotides are preferred, since they are easily
synthesized and are less likely to cause problems than larger
molecules when introduced into the target VLCFA coA-syn-producing
cell. The use of antisense methods to inhibit the in vitro
translation of genes is well known in the art (Marcus-Sakura, Anal.
Biochem., 172:289, 1988).
[0111] Use of an oligonucleotide to stall transcription is known as
the triplex strategy since the oligomer winds around double-helical
DNA, forming a three-strand helix. Therefore, these triplex
compounds can be designed to recognize a unique site on a chosen
gene (Maher, et al., Antisense Res. and Dev., 1:227, 1991; Helene,
Anticancer Drug Design, 6:569, 1991).
[0112] Ribozymes are RNA molecules possessing the ability to
specifically cleave other single-stranded RNA in a manner analogous
to DNA restriction endonucleases. Through the modification of
nucleotide sequences which encode these RNAs, it is possible to
engineer molecules that recognize specific nucleotide sequences in
an RNA molecule and cleave it (Cech, J. Amer. Med. Assn., 260:3030,
1988). A major advantage of this approach is that, because they are
sequence-specific, only mRNAs with particular sequences are
inactivated.
[0113] There are two basic types of ribozymes namely,
tetrahymena-type (Hasselhoff, Nature, 334:585, 1988) and
"hammerhead,"-type. Tetrahymena-type ribozymes recognize sequences
which are four bases in length, while "hammerhead"-type ribozymes
recognize base sequences 11-18 bases in length. The longer the
recognition sequence, the greater the likelihood that the sequence
will occur exclusively in the target mRNA species. Consequently,
hammerhead-type ribozymes are preferable to tetrahymena-type
ribozymes for inactivating a specific mRNA species and 18-based
recognition sequences are preferable to shorter recognition
sequences.
[0114] These and other uses of antisense methods to inhibit the in
vivo translation of genes are well known in the art (e.g., De
Mesmaeker, et al., Curr. Opin. Struct. Biol., 5:343, 1995; Gewirtz,
A. M., et al., Proc. Natl. Acad. Sci. U.S.A., 93:3161, 1996b;
Stein, C. A., Chem. and Biol. 3:319, 1996).
[0115] Delivery of antisense, triplex agents, ribozymes,
competitive inhibitors, double-stranded interferring RNA and the
like can be achieved using a recombinant expression vector such as
a chimeric virus or a colloidal dispersion system or by injection.
Various viral vectors which can be utilized for gene therapy as
taught herein include adenovirus, herpes virus, vaccinia, or,
preferably, an RNA virus such as a retrovirus.
[0116] Preferably, the retroviral vector is a derivative of a
murine or avian retrovirus. Examples of retroviral vectors in which
a single foreign gene can be inserted include, but are not limited
to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma
virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous
Sarcoma Virus (RSV). A number of additional retroviral vectors can
incorporate multiple genes. All of these vectors can transfer or
incorporate a gene for a selectable marker so that transduced cells
can be identified and generated. By inserting a polynucleotide
sequence of interest into the viral vector, along with another gene
which encodes the ligand for a receptor on a specific target cell,
for example, the vector is now target specific. Retroviral vectors
can be made target specific by inserting, for example, a
polynucleotide encoding a sugar, a glycolipid, or a protein.
Preferred targeting is accomplished by using an antibody to target
the retroviral vector. Those of skill in the art will know of, or
can readily ascertain without undue experimentation, specific
polynucleotide sequences which can be inserted into the retroviral
genome to allow target specific delivery of the retroviral vector
containing the antisense polynucleotide.
[0117] Since recombinant retroviruses are defective, they require
assistance in order to produce infectious vector particles. This
assistance can be provided, for example, by using helper cell lines
that contain plasmids encoding all of the structural genes of the
retrovirus under the control of regulatory sequences within the
LTR. These plasmids are missing a nucleotide sequence which enables
the packaging mechanism to recognize an RNA transcript for
encapsidation. Helper cell lines which have deletions of the
packaging signal include but are not limited to T2, PA317 and PA12,
for example. These cell lines produce empty virions, since no
genome is packaged. If a retroviral vector is introduced into such
cells in which the packaging signal is intact, but the structural
genes are replaced by other genes of interest, the vector can be
packaged and vector virion produced.
[0118] Alternatively, NIH 3T3 or other tissue culture cells can be
directly transfected with plasmids encoding the retroviral
structural genes gag, pol and env, by conventional calcium
phosphate transfection. These cells are then transfected with the
vector plasmid containing the genes of interest. The resulting
cells release the retroviral vector into the culture medium.
[0119] Another targeted delivery system for polynucleotides is a
colloidal dispersion system. Colloidal dispersion systems include
macromolecule complexes, nanocapsules, microspheres, beads, and
lipid-based systems including oil-in-water emulsions, micelles,
mixed micelles, and liposomes. The preferred colloidal system of
this invention is a liposome. Liposomes are artificial membrane
vesicles which are useful as delivery vehicles in vitro and in
vivo. It has been shown that large unilamellar vesicles (LUV),
which range in size from 0.2-4.0 .mu.m can encapsulate a
substantial percentage of an aqueous buffer containing large
macromolecules. RNA, DNA and intact virions-can be encapsulated
within the aqueous interior and be delivered to cells in a
biologically active form (Fraley, et al., Trends Biochem. Sci.,
6:77, 1981). In addition to mammalian cells, liposomes have been
used for delivery of polynucleotides in plant, yeast and bacterial
cells. In order for a liposome to be an efficient gene transfer
vehicle, the following characteristics should be present: (1)
encapsulation of the genes of interest at high efficiency while not
compromising their biological activity; (2) preferential and
substantial binding to a target cell in comparison to non-target
cells; (3) delivery of the aqueous contents of the vesicle to the
target cell cytoplasm at high efficiency; and (4) accurate and
effective expression of genetic information (Mannino, et al.,
Biotechniques, 6:682, 1988).
[0120] The composition of the liposome is usually a combination of
phospholipids, particularly high-phase-transition-temperature
phospholipids, usually in combination with steroids, especially
cholesterol. Other phospholipids or other lipids may also be used.
The physical characteristics of liposomes depend on pH, ionic
strength, and the presence of divalent cations.
[0121] Examples of lipids useful in liposome production include
phosphatidyl compounds, such as phosphatidylglycerol,
phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
sphingolipids, cerebrosides, and gangliosides. Particularly useful
are diacylphosphatidyl-glycerols, where the lipid moiety contains
from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and
is saturated. Illustrative phospholipids include egg
phosphatidylcholine, dipalmitoylphosphatidylcholine and
distearoylphosphatidylcholine.
[0122] The targeting of liposomes has been classified based on
anatomical and mechanistic factors. Anatomical classification is
based on the level of selectivity, for example, organ-specific,
cell-specific, and organelle-specific. Mechanistic targeting can be
distinguished based upon whether it is passive or active. Passive
targeting utilizes the natural tendency of liposomes to distribute
to cells of the reticulo-endothelial system (RES) in organs which
contain sinusoidal capillaries. Active targeting, on the other
hand, involves alteration of the liposome by coupling the liposome
to a specific ligand such as a monoclonal antibody, sugar,
glycolipid, or protein, or by changing the composition or size of
the liposome in order to achieve targeting to organs and cell types
other than the naturally occurring sites of localization.
[0123] The surface of the targeted delivery system may be modified
in a variety of ways. In the case of a liposomal targeted delivery
system, lipid groups can be incorporated into the lipid bilayer of
the liposome in order to maintain the targeting ligand in stable
association with the liposomal bilayer. Various linking groups can
be used for joining the lipid chains to the targeting ligand. In
general, the compounds bound to the surface of the targeted
delivery system will be ligands and receptors which will allow the
targeted delivery system to find and "home in" on the desired
cells. A ligand may be any compound of interest which will bind to
another compound, such as a receptor.
[0124] The agents useful in the method of the invention can be
administered, for in vivo application, parenterally by injection or
by gradual perfusion over time. Administration may be
intravenously, intraperitoneally, intramuscularly, subcutaneously,
intracavity, or transdermally. For in vitro studies the agents may
be added or dissolved in an appropriate biologically acceptable
buffer and added to a cell or tissue.
[0125] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's intravenous vehicles include fluid and nutrient
replenishers, electrolyte replenishers (such as those based on
Ringer's dextrose), and the like. Preservatives and other additives
may also be present such as, for example, antimicrobials,
anti-oxidants, chelating agents and inert gases and the like.
[0126] Pharmaceutical Compositions
[0127] It is envisioned that the methods of the present invention
can be used to treat pathologies associated with neurodegeneration.
Therefore, the present invention encompasses methods for
ameliorating a disorder associated with VLCFA coA-syn, including
treating a subject having the disorder, at the site of the
disorder, with a VLCFA coA-syn reactive agent. Generally, the terms
"treating", "treatment" and the like are used herein to mean
affecting a subject, tissue or cell to obtain a desired
pharmacologic and/or physiologic effect. The effect may be
prophylactic in terms of completely or partially preventing a
disease or sign or symptom thereof, and/or may be therapeutic in
terms of a partial or complete cure for an infection or disease
and/or adverse effect attributable to the infection or disease.
"Treating" as used herein covers any treatment of, or prevention
of, an infection or disease in an invertebrate, a vertebrate, a
mammal, particularly a human, and includes:
[0128] (a) preventing the disease from occurring in a subject that
may be predisposed to the disease, but has not yet been diagnosed
as having it;
[0129] (b) inhibiting the disease, i.e., arresting its development;
or
[0130] .COPYRGT. relieving or ameliorating the disease, i.e., cause
regression of the disease.
[0131] However, it should be recognized that the compositions and
methods described herein, can be used to bring about a desired
result (e.g., an increase in life span, increase .beta.-oxidation
of VLCFAs and a decrease in VLCFAs) in the absence of a disease or
disorder.
[0132] Thus, the invention includes various pharmaceutical
compositions useful for ameliorating symptoms attributable to a
VLCFA coA-syn-associated disorder. The pharmaceutical compositions
according to the invention are prepared by bringing an antibody
against VLCFA coA-syn, a polypeptide or peptide derivative of VLCFA
coA-syn, a VLCFA coA-syn mimetic, a drug, chemical or combination
of chemicals (e.g., a combination of oils) or a VLCFA
coA-syn-binding agent according to the present invention into a
form suitable for administration to a subject using carriers,
excipients and additives or auxiliaries. Frequently used carriers
or auxiliaries include magnesium carbonate, titanium dioxide,
lactose, mannitol and other sugars, talc, milk protein, gelatin,
starch, vitamins, cellulose and its derivatives, animal and
vegetable oils, polyethylene glycols and solvents, such as sterile
water, alcohols, glycerol and polyhydric alcohols. Intravenous
vehicles include fluid and nutrient replenishers. Preservatives
include antimicrobial, anti-oxidants, chelating agents and inert
gases. Other pharmaceutically acceptable carriers include aqueous
solutions, non-toxic excipients, including salts, preservatives,
buffers and the like, as described, for instance, in Remington's
Pharmaceutical Sciences, 15th ed. Easton: Mack Publishing Co.,
1405-1412, 1461-1487 (1975) and The National Formulary XIV., 14th
ed. Washington: American Pharmaceutical Association (1975), the
contents of which are hereby incorporated by reference. The pH and
exact concentration of the various components of the pharmaceutical
composition are adjusted according to routine skills in the art.
See Goodman and Gilman's The Pharmacological Basis for Therapeutics
(7th ed.).
[0133] The pharmaceutical compositions are preferably prepared and
administered in dose units. Solid dose units are tablets, capsules
and suppositories. For treatment of a subject, depending on
activity of the compound, manner of administration, nature and
severity of the disorder, age and body weight of the subject,
different daily doses are necessary. Under certain circumstances,
however, higher or lower daily doses may be appropriate. The
administration of the daily dose can be carried out both by single
administration in the form of an individual dose unit or else
several smaller dose units and also by multiple administration of
subdivided doses at specific intervals.
[0134] The pharmaceutical compositions according to the invention
may be administered locally or systemically in a therapeutically
effective dose. By "therapeutically effective dose" is meant the
quantity of a compound according to the invention necessary to
prevent, to cure or at least partially arrest the symptoms of the
disease and its complications. Amounts effective for this use will,
of course, depend on the severity of the disease and the weight and
general state of the subject. Typically, dosages used in vitro may
provide useful guidance in the amounts useful for in situ
administration of the pharmaceutical composition, and animal models
may be used to determine effective dosages for treatment of
particular disorders. Various considerations are described, e.g.,
in Langer, Science, 249: 1527, (1990); Gilman et al. (eds.) (1990),
each of which is herein incorporated by reference.
[0135] In one embodiment, the invention provides a pharmaceutical
composition useful for administering a VLCFA coA-syn polypeptide,
or nucleic acid encoding a VLCFA coA-syn polypeptide, to a subject
in need of such treatment. "Administering" the pharmaceutical
composition of the present invention may be accomplished by any
means known to the skilled artisan. Preferably a "subject" refers
to a mammal, most preferably a human, but may be any organism.
[0136] The VLCFA coA-syn protein or antibody can be administered
parenterally, enterically, by injection, rapid infusion,
nasopharyngeal absorption, dermal absorption, rectally and orally.
Pharmaceutically acceptable carrier preparations for parenteral
administration include sterile or aqueous or non-aqueous solutions,
suspensions, and emulsions. Examples of non-aqueous solvents are
propylene glycol, polyethylene glycol, vegetable oils such as olive
oil, and injectable organic esters such as ethyl oleate. Carriers
for occlusive dressings can be used to increase skin permeability
and enhance antigen absorption. Liquid dosage forms for oral
administration may generally comprise a liposome solution
containing the liquid dosage form. Suitable solid or liquid
pharmaceutical preparation forms are, for example, granules,
powders, tablets, coated tablets, (micro)capsules, suppositories,
syrups, emulsions, suspensions, creams, aerosols, drops or
injectable solution in ampule form and also preparations with
protracted release of active compounds, in whose preparation
excipients and additives and/or auxiliaries such as disintegrants,
binders, coating agents, swelling agents, lubricants, flavorings,
sweeteners and elixirs containing inert diluents commonly used in
the art, such as purified water.
[0137] Screening Assay for Compounds that Affect VLCFA coA-syns
[0138] In another embodiment, the invention provides a method for
identifying a compound which modulates VLCFA coA-syn expression or
activity including incubating components comprising the compound
and a VLCFA coA-syn polypeptide, or a recombinant cell expressing a
VLCFA coA-syn polypeptide, under conditions sufficient to allow the
components to interact and determining the affect of the compound
on the expression or activity of the gene or polypeptide,
respectively. The term "affect", as used herein, encompasses any
means by which VLCFA coA-syn gene expression or protein activity
can be modulated. Such compounds can include, for example,
polypeptides, peptidomimetics, chemical compounds and biologic
agents as described below.
[0139] Incubating includes conditions which allow contact between
the test compound and VLCFA coA-syn, a cell expressing VLCFA
coA-syn or nucleic acid encoding VLCFA coA-syn. Contacting includes
in solution and in solid phase. The test ligand(s)/compound may
optionally be a combinatorial library for screening a plurality of
compounds. Compounds identified in the method of the invention can
be further evaluated, detected, cloned, sequenced, and the like,
either in solution or after binding to a solid support, by any
method usually applied to the detection of a specific DNA sequence
such as PCR, oligomer restriction (Saiki, et al., Bio/Technology,
3:1008-1012, 1985), oligonucleotide ligation assays (OLAs)
(Landegren, et al., Science, 241:1077, 1988), and the like.
Molecular techniques for DNA analysis have been reviewed
(Landegren, et al., Science, 242:229-237, 1988).
[0140] Thus, the method of the invention includes combinatorial
chemistry methods for identifying chemical compounds that bind to
VLCFA coA-syn or affect VLCFA coA-syn expression or activity. By
providing for the production of large amounts of a VLCFA coA-syn,
one can identify ligands or substrates that bind to, modulate,
affect the expression of, or mimic the action of a VLCFA coA-syn.
For example, a polypeptide may have biological activity associated
with the wild-type protein, or may have a loss of function mutation
due to a point mutation in the coding sequence, substitution,
insertion, deletion and scanning mutations.
[0141] Areas of investigation are the development of therapeutic
treatments. The screening identifies agents that provide modulation
of VLCFA coA-syn function in targeted organisms. Of particular
interest are screening assays for agents that have a low toxicity
for humans. A wide variety of assays may be used for this purpose,
including labeled in vitro protein-protein binding assays,
protein-DNA binding assays, electrophoretic mobility shift assays,
immunoassays for protein binding, and the like. The purified
protein may also be used for determination of three-dimensional
crystal structure, which can be used for modeling intermolecular
interactions, for example.
[0142] The term "agent" as used herein describes any molecule, e.g.
protein or pharmaceutical, with the capability of altering or
mimicking the physiological function or expression of a VLCFA
coA-syn. Generally, a plurality of assay mixtures are run in
parallel with different agent concentrations to obtain a
differential response to the various concentrations. Typically, one
of these concentrations serves as a negative control, i.e. at zero
concentration or below the level of detection.
[0143] Candidate agents encompass numerous chemical classes, though
typically they are organic molecules, preferably small organic
compounds having a molecular weight of more than 50 and less than
about 2,500 daltons. Candidate agents comprise functional groups
necessary for structural interaction with proteins, particularly
hydrogen bonding, and typically include at least an amine,
carbonyl, hydroxyl or carboxyl group, preferably at least two of
the functional chemical groups. The candidate agents often comprise
cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more of the above
functional groups. Candidate agents are also found among
biomolecules including, but not limited to: peptides, saccharides,
fatty acids, steroids, purines, pyrimidines, derivatives,
structural analogs or combinations thereof. Candidate agents are
obtained from a wide variety of sources including libraries of
synthetic or natural compounds. For example, numerous means are
available for random and directed synthesis of a wide variety of
organic compounds and biomolecules, including expression of
randomized oligonucleotides and oligopeptides. Alternatively,
libraries of natural compounds in the form of bacterial, fungal,
plant and animal extracts are available or readily produced.
Additionally, natural or synthetically produced libraries and
compounds are readily modified through conventional chemical,
physical and biochemical means, and may be used to produce
combinatorial libraries. Known pharmacological agents may be
subjected to directed or random chemical modifications, such as
acylation, alkylation, esterification and amidification to produce
structural analogs.
[0144] Where the screening assay is a binding assay, one or more of
the molecules may be joined to a label, where the label can
directly or indirectly provide a detectable signal. Various labels
include radioisotopes, fluorescers, chemiluminescers, enzymes,
specific binding molecules, particles, e.g. magnetic particles, and
the like. Specific binding molecules include pairs, such as biotin
and streptavidin, digoxin and antidigoxin. For the specific binding
members, the complementary member would normally be labeled with a
molecule that provides for detection, in accordance with known
procedures.
[0145] A variety of other reagents may be included in the screening
assay. These include reagents like salts, neutral proteins, e.g.
albumin, detergents, etc that are used to facilitate optimal
protein-protein binding and/or reduce non-specific or background
interactions. Reagents that improve the efficiency of the assay,
such as protease inhibitors, nuclease inhibitors and anti-microbial
agents may be used. The mixture of components are added in any
order that provides for the requisite binding. Incubations are
performed at any suitable temperature, typically between 4 and
40.degree. C. Incubation periods are selected for optimum activity,
but may also be optimized to facilitate rapid high-throughput
screening. Typically between 0.1 and 1 hours will be
sufficient.
[0146] In addition, cells or organisms which have a mutation in a
VLCFA-coA synthetase may be use as models to screen for agents
which modulate disorders associated with the mutation. For example,
the inventors have identified that organisms (e.g., Drosophila)
which lack VLCFA coA-synthetase demonstrate a phenotype similar to
ALD resulting in neurodegeneration and a shortened life-span.
Accordingly, administration of agents to organism having such a
mutation, or cells derived or recombinantly modified to have a
reduced VLCFA coA-synthetase activity may be used to determine the
effect of the drug or agent on neurodegeneration.
[0147] Detection of VLCFA coA-syn In Vivo and In Vitro
[0148] In a further embodiment, the invention provides a method of
detecting VLCFA coA-syn or a VLCFA coA-syn-associated disorder in a
subject including contacting a cell component containing VLCFA
coA-syn with a reagent which binds to the cell component.
[0149] The cell component can be nucleic acid, such as DNA or RNA,
or it can be protein. When the component is nucleic acid, the
reagent is a nucleic acid probe or PCR primer. When the cell
component is protein, the reagent is an antibody probe. The probes
are detectably labeled, for example, with a radioisotope, a
fluorescent compound, a bioluminescent compound, a chemiluminescent
compound, a metal chelator or an enzyme. Those of ordinary skill in
the art will know of other labels suitable for binding to an
antibody or nucleic acid probe, or will be able to ascertain such,
using routine experimentation.
[0150] For purposes of the invention, an antibody or nucleic acid
probe specific for VLCFA coA-syn may be used to detect the presence
of VLCFA coA-syn polypeptide (using antibody) or polynucleotide
(using nucleic acid probe) in biological fluids or tissues. Any
specimen containing a detectable amount of VLCFA coA-syn antigen or
polynucleotide can be used. In addition, antibodies and
polynucleotides designed to recognize mutations in a VLCFA
coA-synthetase polypeptide or polynucleotide may be used. For
example, specimens of this invention include blood, urine,
cerebrospinal fluid, synovial fluid or any tissue.
[0151] Another technique which may also result in greater
sensitivity consists of coupling antibodies to low molecular weight
haptens. These haptens can then be specifically detected by means
of a second reaction. For example, it is common to use such haptens
as biotin, which reacts with avidin, or dinitrophenyl, pyridoxal,
and fluorescein, which can react with specific antihapten
antibodies.
[0152] Alternatively, VLCFA coA-syn polypeptide can be used to
detect antibodies to VLCFA coA-syn polypeptide in a specimen. The
VLCFA coA-syn of the invention is particularly suited for use in
immunoassays in which it can be utilized in liquid phase or bound
to a solid phase carrier. In addition, VLCFA coA-syn used in these
assays can be detectably labeled in various ways.
[0153] Examples of immunoassays which can utilize the VLCFA coA-syn
of the invention are competitive and noncompetitive immunoassays in
either a direct or indirect format. Examples of such immunoassays
are the radioimmunoassay (RIA), the sandwich (immunometric assay)
and the Western blot assay. Detection of antibodies which bind to
the VLCFA coA-syn of the invention can be done utilizing
immunoassays which run in either the forward, reverse, or
simultaneous modes, including immunohistochemical assays on
physiological samples. The concentration of VLCFA coA-syn which is
used will vary depending on the type of immunoassay and nature of
the detectable label which is used. However, regardless of the type
of immunoassay which is used, the concentration of VLCFA coA-syn
utilized can be readily determined by one of ordinary skill in the
art using routine experimentation.
[0154] The VLCFA coA-syn of the invention can be bound to many
different carriers and used to detect the presence of antibody
specifically reactive with the polypeptide. Examples of well-known
carriers include glass, polystyrene, polyvinyl chloride,
polypropylene, polyethylene, polycarbonate, dextran, nylon,
amyloses, natural and modified celluloses, polyacrylamides,
agaroses, and magnetite. The nature of the carrier can be either
soluble or insoluble for purposes of the invention.
[0155] Those skilled in the art will know of other suitable
carriers for binding VLCFA coA-syn or will be able to ascertain
such, using routine experimentation.
[0156] There are many different labels and methods of labeling
known to those of ordinary skill in the art. Examples of the types
of labels which can be used in the present invention include
enzymes, radioisotopes, colloidal metals, fluorescent compounds,
chemiluminescent compounds, and bioluminescent compounds.
[0157] For purposes of the invention, the antibody which binds to
VLCFA coA-syn of the invention may be present in various biological
fluids and tissues. Any sample containing a detectable amount of
antibodies to VLCFA coA-syn can be used. Typically, a sample is a
liquid such as urine, saliva, cerebrospinal fluid, blood, serum and
the like, or a solid or semi-solid such as tissue, feces and the
like.
[0158] The monoclonal antibodies of the invention, directed toward
VLCFA coA-syn, are also useful for the in vivo detection of
antigen. The detectably labeled monoclonal antibody is given in a
dose which is diagnostically effective. The term "diagnostically
effective" means that the amount of detectably labeled monoclonal
antibody is administered in sufficient quantity to enable detection
of VLCFA coA-syn antigen for which the monoclonal antibodies are
specific.
[0159] The concentration of detectably labeled monoclonal antibody
which is administered should be sufficient such that the binding to
those cells, body fluid, or tissue having VLCFA coA-syn is
detectable compared to the background. Further, it is desirable
that the detectably labeled monoclonal antibody be rapidly cleared
from the circulatory system in order to give the best
target-to-background signal ratio.
[0160] For in vivo diagnostic imaging, the type of detection
instrument available is a major factor in selecting a given
radioisotope. The radioisotope chosen must have a type of decay
which is detectable for a given type of instrument. Still another
important factor in selecting a radioisotope for in vivo diagnosis
is that the half-life of the radioisotope be long enough so that it
is still detectable at the time of maximum uptake by the target,
but short enough so that deleterious radiation with respect to the
host is minimized. Ideally, a radioisotope used for in vivo imaging
will lack a particle emission, but produce a large number of
photons in the 140-250 key range, which may be readily detected by
conventional gamma cameras.
[0161] For in vivo diagnosis, radioisotopes may be bound to
immunoglobulin either directly or indirectly by using an
intermediate functional group. Intermediate functional groups which
often are used to bind radioisotopes which exist as metallic ions
to immunoglobulins are the bifunctional chelating agents such as
diethylenetriaminepentacetic acid (DTPA) and
ethylenediaminetetraacetic acid (EDTA) and similar molecules.
Typical examples of metallic ions which can be bound to the
monoclonal antibodies of the invention are .sup.111In, .sup.97Ru,
.sup.67Ga, .sup.68Ga, .sup.72As, .sup.89Zr, and .sup.201Tl.
[0162] The monoclonal antibodies of the invention can also be
labeled with a paramagnetic isotope for purposes of in vivo
diagnosis, as in magnetic resonance imaging (MRI) or electron spin
resonance (ESR). In general, any conventional method for
visualizing diagnostic imaging can be utilized. Usually gamma and
positron emitting radioisotopes are used for camera imaging and
paramagnetic isotopes for MRI. Elements which are particularly
useful in such techniques include .sup.157Gd, .sup.55Mn,
.sup.162Dy, .sup.52Cr, and .sup.56Fe.
[0163] The monoclonal antibodies of the invention can be used to
monitor the course of amelioration of a VLCFA coA-syn-associated
disorder. Thus, by measuring the increase or decrease of VLCFA
coA-syn polypeptide present in various body fluids or tissues, it
would be possible to determine whether a particular therapeutic
regiment aimed at ameliorating the disorder is effective.
[0164] In another embodiment, nucleic acid probes can be used to
identify VLCFA coA-syn nucleic acid from a specimen obtained from a
subject. Examples of specimens from which nucleic acid sequence
encoding VLCFA coA-syn can be derived include insect, human, swine,
porcine, feline, canine, equine, murine, cervine, caprine, lupine,
leporidine and bovine species.
[0165] Oligonucleotide probes, which correspond to a part of the
sequence encoding the protein in question, can be synthesized
chemically. This requires that short, oligopeptide stretches of
amino acid sequence must be known. The DNA sequence encoding the
protein can be deduced from the genetic code, however, the
degeneracy of the code must be taken into account. It is possible
to perform a mixed addition reaction when the sequence is
degenerate. This includes a heterogeneous mixture of denatured
double-stranded DNA. For such screening, hybridization is
preferably performed on either single-stranded DNA or denatured
double-stranded DNA. Hybridization is particularly useful in the
detection of cDNA clones derived from sources where an extremely
low amount of mRNA sequences relating to the polypeptide of
interest are present. In other words, by using stringent
hybridization conditions directed to avoid non-specific binding, it
is possible, for example, to allow the autoradiographic
visualization of a specific cDNA clone by the hybridization of the
target DNA to that single probe in the mixture which is its
complete complement (Wallace, et al., Nucl. Acid Res. 9:879,
1981).
[0166] In an embodiment of the invention, purified nucleic acid
fragments containing intervening sequences or oligonucleotide
sequences of 10-50 base pairs are radioactively labeled. The
labeled preparations are used to probe nucleic acid from a specimen
by the Southern hybridization technique. Nucleotide fragments from
a specimen, before or after amplification, are separated into
fragments of different molecular masses by gel electrophoresis and
transferred to filters that bind nucleic acid. After exposure to
the labeled probe, which will hybridize to nucleotide fragments
containing target nucleic acid sequences, binding of the
radioactive probe to target nucleic acid fragments is identified by
autoradiography (see Genetic Engineering, 1, ed. Robert Williamson,
Academic Press, (1981), 72-81). Alternatively, nucleic acid from
the specimen can be bound directly to filters to which the
radioactive probe selectively attaches by binding nucleic acids
having the sequence of interest. Specific sequences and the degree
of binding is quantitated by directly counting the radioactive
emissions.
[0167] Where the target nucleic acid is not amplified, detection
using an appropriate hybridization probe may be performed directly
on the separated nucleic acid. In those instances where the target
nucleic acid is amplified, detection with the appropriate
hybridization probe would be performed after amplification.
[0168] For the most part, the probe will be detectably labeled with
an atom or inorganic radical, most commonly using radionuclides,
but also heavy metals can be used. Conveniently, a radioactive
label may be employed. Radioactive labels include .sup.32P,
.sup.125I, .sup.3H, .sup.14C, .sup.111In, .sup.99mTc, or the like.
Any radioactive label may be employed which provides for an
adequate signal and has sufficient half-life. Other labels include
ligands, which can serve as a specific binding pair member for a
labeled ligand, and the like. A wide variety of labels routinely
employed in immunoassays can readily be employed in the present
assay. The choice of the label will be governed by the effect of
the label on the rate of hybridization and binding of the probe to
mutant nucleotide sequence. It will be necessary that the label
provide sufficient sensitivity to detect the amount of mutant
nucleotide sequence available for hybridization. Other
considerations will be ease of synthesis of the probe, readily
available instrumentation, ability to automate, convenience, and
the like.
[0169] The manner in which the label is bound to the probe will
vary depending upon the nature of the label. For a radioactive
label, a wide variety of techniques can be employed. Commonly
employed is nick translation with an a .sup.32P-DNTP or terminal
phosphate hydrolysis with alkaline phosphatase followed by labeling
with radioactive .sup.32P employing .sup.32P-NTP and T4
polynucleotide kinase. Alternatively, nucleotides can be
synthesized where one or more of the elements present are replaced
with a radioactive isotope, e.g., hydrogen with tritium. If
desired, complementary labeled strands can be used as probes to
enhance the concentration of hybridized label.
[0170] Where other radionucleotide labels are involved, various
linking groups can be employed. A terminal hydroxyl can be
esterified, with inorganic acids, e.g., .sup.32P phosphate, or
.sup.14C organic acids, or else esterified to provide linking
groups to the label. Alternatively, intermediate bases may be
substituted with activatable linking groups that can then be linked
to a label.
[0171] Enzymes of interest as reporter groups will primarily be
hydrolases, particularly esterases and glycosidases, or
oxidoreductases, particularly peroxidases. Fluorescent compounds
include fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, umbelliferone, and so forth. Chemiluminescers
include luciferin, and 2,3-dihydrophthalazinediones (e.g.,
luminol).
[0172] The probe can be employed for hybridizing to a nucleotide
sequence affixed to a water insoluble porous support. Depending
upon the source of the nucleic acid, the manner in which the
nucleic acid is affixed to the support may vary. Those of ordinary
skill in the art know, or can easily ascertain, different supports
that can be used in the method of the invention.
[0173] The nucleic acid from a specimen can be cloned and then
spotted or spread onto a filter to provide a plurality of
individual portions (plaques). The filter is an inert porous solid
support, e.g., nitrocellulose. Any cells (or phage) present in the
specimen are treated to liberate their nucleic acid. The lysing and
denaturation of nucleic acid, as well as the subsequent washings,
can be achieved with an appropriate solution for a sufficient time
to lyse the cells and denature the nucleic acid. For lysing,
chemical lysing will conveniently be employed, as described
previously for the lysis buffer. Other denaturation agents include
elevated temperatures, organic reagents, e.g., alcohols, amides,
amines, ureas, phenols and sulfoxides or certain inorganic ions,
e.g., thiocyanate and perchlorate.
[0174] After denaturation, the filter is washed in an aqueous
buffered solution, such as Tris, generally at a pH of about 6 to 8,
usually 7. One or more washings may be involved, conveniently using
the same procedure as employed for the lysing and denaturation.
After the lysing, denaturing, and washes have been accomplished,
the nucleic acid spotted filter is dried at an elevated
temperature, generally from about 50.degree. C. to 70.degree. C.
Under this procedure, the nucleic acid is fixed in position and can
be assayed with the probe when convenient.
[0175] Pre-hybridization may be accomplished by incubating the
filter with the hybridization solution without the probe at a
mildly elevated temperature for a sufficient time to thoroughly wet
the filter. Various hybridization solutions may be employed,
comprising from about 20% to 60% volume, preferably 30%, of an
inert polar organic solvent. A common hybridization solution
employs about 50% formamide, about 0.5 to 1M sodium chloride, about
0.05 to 0.1M sodium citrate, about 0.05 to 0.2% sodium
dodecylsulfate, and minor amounts of EDTA, ficoll (about 300-500
kDa), polyvinylpyrrolidone, (about 250-500 kDa) and serum albumin.
Also included in the hybridization solution will generally be from
about 0.5 to 5 mg/ml of sonicated denatured DNA, e.g., calf thymus
of salmon sperm; and optionally from about 0.5 to 2% wt/vol
glycine. Other additives may also be included, such as dextran
sulfate of from about 100 to 1,000 kDa and in an amount of from
about 8 to 15 weight percent of the hybridization solution.
[0176] The particular hybridization technique is not essential to
the invention. Other hybridization techniques are described by Gall
and Pardue, (Proc. Natl. Acad. Sci. 63:378, 1969); and John, et
al., (Nature, 223:582, 1969). As improvements are made in
hybridization techniques they can readily be applied in the method
of the invention.
[0177] The amount of labeled probe present in the hybridization
solution will vary widely, depending upon the nature of the label,
the amount of the labeled probe that can reasonably bind to the
filter, and the stringency of the hybridization. Generally,
substantial excess over stoichiometric concentrations of the probe
will be employed to enhance the rate of binding of the probe to the
fixed target nucleic acid.
[0178] In nucleic acid hybridization reactions, the conditions used
to achieve a particular level of stringency will vary, depending on
the nature of the nucleic acids being hybridized. For example, the
length, degree of complementarity, nucleotide sequence compound
(e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA)
of the hybridizing regions of the nucleic acids can be considered
in selecting hybridization conditions. An additional consideration
is whether one of the nucleic acids is immobilized, for example, on
a filter.
[0179] After the filter has been contacted with a hybridization
solution at a moderate temperature for a period of time sufficient
to allow hybridization to occur, the filter is then introduced into
a second solution having analogous concentrations of sodium
chloride, sodium citrate and sodium dodecylsulfate as provided in
the hybridization solution. The time the filter is maintained in
the second solution may vary from five minutes to three hours or
more. The second solution determines the stringency, dissolving
cross duplexes and short complementary sequences. After rinsing the
filter at room temperature with dilute sodium citrate-sodium
chloride solution, the filter may now be assayed for the presence
of duplexes in accordance with the nature of the label. Where the
label is radioactive, the filter is dried and exposed to X-ray
film.
[0180] The label may also comprise a fluorescent moiety that can
then be probed with a specific fluorescent antibody. Horseradish
peroxidase enzyme can be conjugated to the antibody to catalyze a
chemiluminescent reaction. Production of light can then be seen on
rapid exposure to film.
[0181] Transgenic Organisms
[0182] The present invention also contemplates transgenic non-human
organisms, including invertebrates, vertebrates and mammals. For
purposes of the subject invention, these animals are referred to as
"transgenic" when such animal has had a heterologous DNA sequence,
or one or more additional DNA sequences normally endogenous to the
animal (collectively referred to herein as "transgenes")
chromosomally integrated into the germ cells of the animal. The
transgenic animal (including its progeny) will also have the
transgene integrated into the chromosomes of somatic cells.
[0183] Various methods to make the transgenic animals of the
subject invention can be employed. Generally speaking, three such
methods may be employed. In one such method, an embryo at the
pronuclear stage (a "one cell embryo") is harvested from a female
and the transgene is microinjected into the embryo, in which case
the transgene will be chromosomally integrated into both the germ
cells and somatic cells of the resulting mature animal. In another
such method, embryonic stem cells are isolated and the transgene
incorporated therein by electroporation, plasmid transfection or
microinjection, followed by reintroduction of the stem cells into
the embryo where they colonize and contribute to the germ line.
Methods for microinjection of mammalian species is described in
U.S. Pat. No. 4,873,191. In yet another such method, embryonic
cells are infected with a retrovirus containing the transgene
whereby the germ cells of the embryo have the transgene
chromosomally integrated therein. When the animals to be made
transgenic are avian, because avian fertilized ova generally go
through cell division for the first twenty hours in the oviduct,
microinjection into the pronucleus of the fertilized egg is
problematic due to the inaccessibility of the pronucleus.
Therefore, of the methods to make transgenic animals described
generally above, retrovirus infection is preferred for avian
species, for example as described in U.S. Pat. No. 5,162,215. If
microinjection is to be used with avian species, however, a
published procedure by Love et al., (Biotechnology, 12, January
1994) can be utilized whereby the embryo is obtained from a
sacrificed hen approximately two and one-half hours after the
laying of the previous laid egg, the transgene is microinjected
into the cytoplasm of the germinal disc and the embryo is cultured
in a host shell until maturity. When the animals to be made
transgenic are bovine or porcine, microinjection can be hampered by
the opacity of the ova thereby making the nuclei difficult to
identify by traditional differential interference-contrast
microscopy. To overcome this problem, the ova can first be
centrifuged to segregate the pronuclei for better
visualization.
[0184] The "non-human animals" of the invention include, for
example, bovine, porcine, ovine and avian animals (e.g., cow, pig,
sheep, chicken, turkey). The "transgenic non-human animals" of the
invention are produced by introducing "transgenes" into the
germline of the non-human animal. Embryonal target cells at various
developmental stages can be used to introduce transgenes. Different
methods are used depending on the stage of development of the
embryonal target cell. The zygote is the best target for
micro-injection. The use of zygotes as a target for gene transfer
has a major advantage in that in most cases the injected DNA will
be incorporated into the host gene before the first cleavage
(Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442, 1985).
As a consequence, all cells of the transgenic non-human animal will
carry the incorporated transgene. This will in general also be
reflected in the efficient transmission of the transgene to
offspring of the founder since 50% of the germ cells will harbor
the transgene.
[0185] The term "transgenic" is used to describe an animal which
includes exogenous genetic material within all of its cells. A
"transgenic" animal can be produced by cross-breeding two chimeric
animals which include exogenous genetic material within cells used
in reproduction. Twenty-five percent of the resulting offspring
will be transgenic i.e., animals which include the exogenous
genetic material within all of their cells in both alleles. 50% of
the resulting animals will include the exogenous genetic material
within one allele and 25% will include no exogenous genetic
material.
[0186] In the microinjection method useful in the practice of the
subject invention, the transgene is digested and purified free from
any vector DNA e.g. by gel electrophoresis. It is preferred that
the transgene include an operatively associated promoter which
interacts with cellular proteins involved in transcription,
ultimately resulting in constitutive expression. Promoters useful
in this regard include those from cytomegalovirus (CMV), Moloney
leukemia virus (MLV), and herpes virus, as well as those from the
genes encoding metallothionin, skeletal actin, P-enolpyruvate
carboxylase (PEPCK), phosphoglycerate (PGK), DHFR, and thymidine
kinase. Promoters for viral long terminal repeats (LTRS) such as
Rous Sarcoma Virus can also be employed. When the animals to be
made transgenic are avian, preferred promoters include those for
the chicken-globin gene, chicken lysozyme gene, and avian leukosis
virus. Constructs useful in plasmid transfection of embryonic stem
cells will employ additional regulatory elements well known in the
art such as enhancer elements to stimulate transcription, splice
acceptors, termination and polyadenylation signals, and ribosome
binding sites to permit translation.
[0187] Retroviral infection can also be used to introduce transgene
into a non-human animal, as described above. The developing
non-human embryo can be cultured in vitro to the blastocyst stage.
During this time, the blastomeres can be targets for retro viral
infection (Jaenich, R., Proc. Natl. Acad. Sci USA 73:1260-1264,
1976). Efficient infection of the blastomeres is obtained by
enzymatic treatment to remove the zona pellucida (Hogan, et al.
(1986) in Manipulating the Mouse Embryo, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.). The viral vector
system used to introduce the transgene is typically a
replication-defective retro virus carrying the transgene (Jahner,
et al., Proc. Natl. Acad. Sci. USA 82:6927-6931, 1985; Van der
Putten, et al., Proc. Natl. Acad. Sci USA 82:6148-6152, 1985).
Transfection is easily and efficiently obtained by culturing the
blastomeres on a monolayer of virus-producing cells (Van der
Putten, supra; Stewart, et al., EMBO J. 6:383-388, 1987).
Alternatively, infection can be performed at a later stage. Virus
or virus-producing cells can be injected into the blastocoele (D.
Jahner et al., Nature 298:623-628, 1982). Most of the founders will
be mosaic for the transgene since incorporation occurs only in a
subset of the cells which formed the transgenic nonhuman animal.
Further, the founder may contain various retro viral insertions of
the transgene at different positions in the genome which generally
will segregate in the offspring. In addition, it is also possible
to introduce transgenes into the germ line, albeit with low
efficiency, by intrauterine retroviral infection of the
midgestation embryo (D. Jahner et al., supra).
[0188] A third type of target cell for transgene introduction is
the embryonal stem cell (ES). ES cells are obtained from
pre-implantation embryos cultured in vitro and fused with embryos
(M. J. Evans et al. Nature 292:154-156, 1981; M. O. Bradley et al.,
Nature 309: 255-258, 1984; Gossler, et al., Proc. Natl. Acad. Sci
USA 83: 9065-9069, 1986; and Robertson et al., Nature 322:445-448,
1986). Transgenes can be efficiently introduced into the ES cells
by DNA transfection or by retro virus-mediated transduction. Such
transformed ES cells can thereafter be combined with blastocysts
from a nonhuman animal. The ES cells thereafter colonize the embryo
and contribute to the germ line of the resulting chimeric animal.
(For review see Jaenisch, R., Science 240: 1468-1474, 1988).
[0189] "Transformed" means a cell into which (or into an ancestor
of which) has been introduced, by means of recombinant nucleic acid
techniques, a heterologous nucleic acid molecule. "Heterologous"
refers to a nucleic acid sequence that either originates from
another species or is modified from either its original form or the
form primarily expressed in the cell.
[0190] "Transgene" means any piece of DNA which is inserted by
artifice into a cell, and becomes part of the genome of the
organism (i.e., either stably integrated or as a stable
extrachromosomal element) which develops from that cell. Such a
transgene may include a gene which is partly or entirely
heterologous (i.e., foreign) to the transgenic organism, or may
represent a gene homologous to an endogenous gene of the organism.
Included within this definition is a transgene created by the
providing of an RNA sequence which is transcribed into DNA and then
incorporated into the genome. The transgenes of the invention
include DNA sequences which encode VLCFA coA-syn, and include VLCFA
coA-syn-sense, antisense, dominant negative encoding
polynucleotides, which may be expressed in a transgenic non-human
animal. The term "transgenic" as used herein additionally includes
any organism whose genome has been altered by in vitro manipulation
of the early embryo or fertilized egg or by any transgenic
technology to induce a specific gene knockout (i.e., knockout of
VLCFA coA-synthetase). The term "gene knockout" as used herein,
refers to the targeted disruption of a gene in vivo with complete
or partial loss of function that has been achieved by any
transgenic technology familiar to those in the art (e.g., insertion
of a P-element in Drosophila). In one embodiment, transgenic
animals having gene knockouts are those in which the target gene
has been rendered nonfunctional by an insertion targeted to the
gene to be rendered non-functional by homologous recombination. As
used herein, the term "transgenic" includes any transgenic
technology familiar to those in the art which can produce an
organism carrying an introduced transgene or one in which an
endogenous gene has been rendered non-functional or "knocked
out."
[0191] In one embodiment, the transgene comprises DNA antisense to
the coding sequence for VLCFA coA-syn. In another embodiment, the
transgene comprises DNA encoding an antibody which is able to bind
to VLCFA coA-syn. Where appropriate, DNA sequences that encode
proteins having VLCFA coA-syn activity but differ in nucleic acid
sequence due to the degeneracy of the genetic code may also be used
herein, as may truncated forms, allelic variants and interspecies
homologues.
[0192] The invention also includes animals having heterozygous
mutations in VLCFA coA-syn or partial inhibition of VLCFA coA-syn
function or expression. Partial loss of function leads to a
decrease in .beta.-oxidation of VLCFAs resulting in a phenotype
characteristic of ALD (e.g., neurodegeneration and shortened
lifespan). One of skill in the art would readily be able to
determine if a particular mutation or if an antisense molecule was
able to partially inhibit VLCFA coA-syn. For example, in vitro
testing may be desirable initially by comparison with wild-type or
untreated VLCFA coA-syn (e.g., comparison of northern blots to
examine a decrease in expression).
[0193] After an embryo has been microinjected, colonized with
transfected embryonic stem cells or infected with a retrovirus
containing the transgene (except for practice of the subject
invention in avian species which is addressed elsewhere herein) the
embryo is implanted into the oviduct of a pseudopregnant female.
The consequent progeny are tested for incorporation of the
transgene by Southern blot analysis of blood samples using
transgene specific probes. PCR is particularly useful in this
regard. Positive progeny (G0) are crossbred to produce offspring
(G1) which are analyzed for transgene expression by Northern blot
analysis of tissue samples. To be able to distinguish expression of
like-species transgenes from expression of the animals endogenous
VLCFA coA-syn gene(s), a marker gene fragment can be included in
the construct in the 3' untranslated region of the transgene and
the Northern probe designed to probe for the marker gene fragment.
The serum levels of VLCFA coA-syn can also be measured in the
transgenic animal to establish appropriate expression. Expression
of the VLCFA coA-syn transgenes, thereby decreasing the VLCFA
coA-syn in the tissue and serum levels of the transgenic
animals.
[0194] Transgenic organisms of the invention are highly useful in
the production of organisms for study of neurodegeneration and in
identifying agents or drugs with inhibit or modulate
neurodegeneration (e.g., ALD).
[0195] It will be recognized that the method of creating a
transgenic organism include methods of inserting a transgene into,
for example, an embryo of an already created transgenic organism,
the organism being transgenic for a different unrelated gene or
gene product.
[0196] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following examples are
to be considered illustrative and thus are not limiting of the
remainder of the disclosure in any way whatsoever.
EXAMPLES
[0197] Mutagenesis and screen. Drosophilia lines carrying P(lacZ,
w+) were screened for mutations causing reduced lifespan at
29.degree. C. Candidates showing reduced lifespan compared with the
parent strain were examined after aging, but before death, to
identify those with brain degeneration bubblegum showed early death
associated with degeneration.
[0198] To identify the affected gene in bubblegum, genomic DNA
adjacent to P-element insertion was isolated by plasmid rescue and
sequenced. The genomic sequence of the region had been determined
by the Berkeley genome project and a cDNA clone for the gene was
commercially available. The P-element was found to be inserted into
the region between the 5'UTR and the start codon of an open reading
frame of 2502 bp predicting a protein of 649 amino acids (FIGS.
3A1, 3A2 and 3A3). A Blast protein data bank search indicated
homology with very long chain fatty acid acyl CoA synthetase in the
rat (FIGS. 3B1 and 3B2). The overall similarity between the two
proteins is 53%. A human sequence closely similar to the rat was
also identified (FIGS. 3B1 and 3B2).
[0199] Tissue preparation for microscopy. Fly heads were prepared
by fixation in 1% paraformaldehyde+1% glutaraldehyde, postfixation
in 1% osmium tetroxide, dehydration in an ethanol series, and
embedding in Epon 812. For light microscopy, 1 .mu.m series were
stained with 1% toludine blue 1% Borax. For electron microscopy,
ultrathin sections (80 nm) were examined with a Philips 201
electron microscope at 60 kV.
[0200] Molecular cloning of the bubblegum gene. Genomic DNA
sequences adjacent to the P-element insertion were identified via
data from the Drosophila Berkeley genome project. The cDNA clone
containing the bubblegum gene was purchased from Genome Systems. A
single insertion of the P-element was confirmed by genomic Southern
blots. Precise excision of the P-element from the mutant restored
the normal phenotype.
[0201] Assay of very long chain fatty acids by gas
chromatography.
[0202] To determine whether bubblegum has an accumulation of
VLCFAs, fatty acid analysis by gas chromatography was performed on
15 day old flies, comparing homozygous mutant flies with those of
the parent strain from which bubblegum was derived. Indeed, the
mutant males showed increased levels of VLCFAs, including C22, C24,
and C26 whereas those of chain length below C20 did not increase
(FIG. 4).
[0203] Extracts of whole male or female flies were used for
purification and methylation of fatty acids. Gas chromatography was
performed with a HP-5MS column (30m.times.0.25 mm, 0.25-pm film
thickness) and mass spectrometry was used to analyze the fatty acid
methyl esters. Identification and measurement of fatty acid
concentrations were based on an oil reference standard (NHI-F) from
Sigma Co. and the C15 added as an internal standard.
[0204] To determine whether similar dietary treatment can reduce
the level of C26 and/or prevent the pathology seen in bubblegum,
2.5% of GTO oil was added to the normal corn meal-yeast-agar
medium. When adult mutant flies were transferred to that medium,
the onset of brain degeneration was reduced only slightly; the
photoreceptor axons in the lamina still expanded (FIG. 5a).
However, knowing that oil treatment in ALD was not able to prevent
the progression of the disease once patients had shown neurological
symptoms, we decided to treat mutant flies with the oil at
pre-adult stages. First instar larvae were raised in the
oil-containing medium until eclosion and the adult flies were then
transferred into fresh medium, still containing the oil, changed
every 3 days. Both light microscopy and electron microscopy of 15
day-old adult flies revealed that the degeneration in the
oil-treated mutant flies had largely been prevented (FIG. 5b). In
addition, the levels of VLCFAs were reduced to normal (FIG. 4b),
and the lifespan recovered. The oil had no evident effects on flies
with the normal gene.
[0205] Phototactic analysis. In ALD, visual loss is on of the
clinical symptoms, and anatomical analysis has shown abnormalities
of the optic nerve and degeneration of ganglion cell layer. Flies
transformed with P-elements expressing eye pigment, as is the case
for bubblegum, are phototactic. A countercurrent phototaxis test
was used to examine the visual behavior of bubblegum. While mutant
flies, raise in ordinary medium, showed little response, those
raised with the oil from the larval stage showed a phototactic
response, as seen in FIG. 5c. Therefore, the oil treatment can
prevent the biochemical, structural, and physiological defects
caused by the mutation.
[0206] Flies were placed in a countercurrent distribution apparatus
and responses to light were analyzed. Each numbered tube represents
the number of positive responses in a total of 5 trials. In control
tests for the from light response, neither group of flies moved
away from light.
[0207] The data given above for bubblegum refer to male flies
homozygous for the second-chromosome P-element insertion.
Homozygous females show the same age-dependent degeneration
phenotype, which is similarly prevented by feeding the oil. It was
therefore surprising to find that the excess of VLCFAs seen in
males was not present in females analyzed by the same procedure.
Nor did females show a reduction in lifespan. These aberrations
suggest that there might not be a direct causal linkage between the
fatty acid anomaly and the bubblegum phenotype, which may, instead,
be separate ramifications of an underlying defect, with females
being able to compensate for some, but not all, of the
consequences. This paradox prompts further inquiry. Whether a
similar effect occurs with the X-linked ALD gene in humans is very
difficult to determine, since the occurrence of homozygous females
is vanishing rare.
Sequence CWU 1
1
4 1 2502 DNA Drosophila melanogaster CDS (161)...(2107) 1
ctgaattcgg tcgtgtttgc tgtcgtggtt ctcgagcgaa agaaagagtg ggagtataga
60 aaatagacgg caatcgattt gcgtgaccaa agaacaaata tatacataca
tatatcgaga 120 acgccgtaga aacaccaaac tagttaatta tccttgcaac atg tcc
acg ata gac 175 Met Ser Thr Ile Asp 1 5 gcg ctc tac aat cgt cct ggg
ccc aac cgc ctg cgg cag gcg gat gcc 223 Ala Leu Tyr Asn Arg Pro Gly
Pro Asn Arg Leu Arg Gln Ala Asp Ala 10 15 20 tat cgc acc acc aat
cgt cag gat gcc gtc aag att cgt atg gcc aag 271 Tyr Arg Thr Thr Asn
Arg Gln Asp Ala Val Lys Ile Arg Met Ala Lys 25 30 35 gat gga atc
ggc gca gag gag ccc atc tcc gtg ccc ggc ctg ctg aag 319 Asp Gly Ile
Gly Ala Glu Glu Pro Ile Ser Val Pro Gly Leu Leu Lys 40 45 50 cgt
acg gtc aac aat tat ggc gac tat cct gcg ctg cgc acc aag aac 367 Arg
Thr Val Asn Asn Tyr Gly Asp Tyr Pro Ala Leu Arg Thr Lys Asn 55 60
65 ggc aag aac gga tat cac act gtc acc tac aaa caa tat gag cag aag
415 Gly Lys Asn Gly Tyr His Thr Val Thr Tyr Lys Gln Tyr Glu Gln Lys
70 75 80 85 gtg cac cag gtg gcc aag gcg ttc att aag ctc ggt ctg gag
gag cac 463 Val His Gln Val Ala Lys Ala Phe Ile Lys Leu Gly Leu Glu
Glu His 90 95 100 cat tcg gtg ggt gtg ctg gcc ttc aat tgc gcc gaa
tgg ttc tac tcg 511 His Ser Val Gly Val Leu Ala Phe Asn Cys Ala Glu
Trp Phe Tyr Ser 105 110 115 gcc atg ggc gcc att cac gca cga ggc atc
atc gcc gga atc tac acc 559 Ala Met Gly Ala Ile His Ala Arg Gly Ile
Ile Ala Gly Ile Tyr Thr 120 125 130 acc aat tcc gcc gat gca gtg cag
cac gtt ctg gag agc tca cat gcc 607 Thr Asn Ser Ala Asp Ala Val Gln
His Val Leu Glu Ser Ser His Ala 135 140 145 caa atc gtg gtc gtc gac
gac gcc aag caa atg gac aag att cac gcc 655 Gln Ile Val Val Val Asp
Asp Ala Lys Gln Met Asp Lys Ile His Ala 150 155 160 165 att cgc gac
aag ctg ccc aag ctc aag gcc gcc att cag atc cag gag 703 Ile Arg Asp
Lys Leu Pro Lys Leu Lys Ala Ala Ile Gln Ile Gln Glu 170 175 180 ccc
tat tcc ccc tac ttg aag aag gag gat ggc tac tac agg tgg tcg 751 Pro
Tyr Ser Pro Tyr Leu Lys Lys Glu Asp Gly Tyr Tyr Arg Trp Ser 185 190
195 gag atc gag tcg atg aac gtt agc gac gtg gag gat cag tac atg acc
799 Glu Ile Glu Ser Met Asn Val Ser Asp Val Glu Asp Gln Tyr Met Thr
200 205 210 cgt ttg gag aat gtg gcg atc aac gag tgc tgc tgc ctg gtc
tac acc 847 Arg Leu Glu Asn Val Ala Ile Asn Glu Cys Cys Cys Leu Val
Tyr Thr 215 220 225 tcc gga acg gtg ggc atg ccc aag ggc gtg atg ctc
tcc cac gac aac 895 Ser Gly Thr Val Gly Met Pro Lys Gly Val Met Leu
Ser His Asp Asn 230 235 240 245 atc acc ttc gat gtg cgc ggc atc gtc
aag gcc atg gac cgt gtg gtg 943 Ile Thr Phe Asp Val Arg Gly Ile Val
Lys Ala Met Asp Arg Val Val 250 255 260 gtt ggg gcg gag tcg atc gtc
tcc tac ctg cca ctt tcg cac gtg gcc 991 Val Gly Ala Glu Ser Ile Val
Ser Tyr Leu Pro Leu Ser His Val Ala 265 270 275 gcc cag acc gtg gac
att tac acc tgc gcc ttt gtg gcg ggc tgc att 1039 Ala Gln Thr Val
Asp Ile Tyr Thr Cys Ala Phe Val Ala Gly Cys Ile 280 285 290 tgg ttc
gcc gac aag gat gcg ctg aag gga acg ctg gtg aag tcg ttg 1087 Trp
Phe Ala Asp Lys Asp Ala Leu Lys Gly Thr Leu Val Lys Ser Leu 295 300
305 cag gat gcg cga ccc acg cga ttc atg ggc gtg ccg cgt gtg tac gag
1135 Gln Asp Ala Arg Pro Thr Arg Phe Met Gly Val Pro Arg Val Tyr
Glu 310 315 320 325 aag ttc cag gag cga atg gtc gcc gtg gcc agc tcc
agc ggc agc ctg 1183 Lys Phe Gln Glu Arg Met Val Ala Val Ala Ser
Ser Ser Gly Ser Leu 330 335 340 aag aag atg ctc gcc agc tgg gcc aag
ggc atc acg ctg aag cac tac 1231 Lys Lys Met Leu Ala Ser Trp Ala
Lys Gly Ile Thr Leu Lys His Tyr 345 350 355 atg gtg agt caa ggc aag
agc tcc ggg gga ttc cgg tac aag att gcc 1279 Met Val Ser Gln Gly
Lys Ser Ser Gly Gly Phe Arg Tyr Lys Ile Ala 360 365 370 aag tcg ctc
atc atg tcc aag gtg aag cag gcc ctg ggc ttc gat cgc 1327 Lys Ser
Leu Ile Met Ser Lys Val Lys Gln Ala Leu Gly Phe Asp Arg 375 380 385
gtc ctt aca ctg gcc agt gcg gca gct ccc atg tcg ccg gag acg aag
1375 Val Leu Thr Leu Ala Ser Ala Ala Ala Pro Met Ser Pro Glu Thr
Lys 390 395 400 405 aag tac ttc ctc agt ctg gac cta aag att gtc gat
gcc ttc ggc atg 1423 Lys Tyr Phe Leu Ser Leu Asp Leu Lys Ile Val
Asp Ala Phe Gly Met 410 415 420 tca gaa acg gcc ggt tgt cac acc atc
tgc ctt ccc gat tcc gtg ggt 1471 Ser Glu Thr Ala Gly Cys His Thr
Ile Cys Leu Pro Asp Ser Val Gly 425 430 435 ctg aac aca atc ggc aaa
act ttg ccc ggc tgc gag tcc aag ttc atc 1519 Leu Asn Thr Ile Gly
Lys Thr Leu Pro Gly Cys Glu Ser Lys Phe Ile 440 445 450 aac aag gat
gcc aac ggt cac gga gag ctg tgc atc cga gga cgt cac 1567 Asn Lys
Asp Ala Asn Gly His Gly Glu Leu Cys Ile Arg Gly Arg His 455 460 465
gtt ttc atg ggc tac atc gac aac aag gag aag acc gag gag tcg ctg
1615 Val Phe Met Gly Tyr Ile Asp Asn Lys Glu Lys Thr Glu Glu Ser
Leu 470 475 480 485 gat gac gac tgc tgg ctg cat tcc ggt gat ttg gga
ttt gtg gat gac 1663 Asp Asp Asp Cys Trp Leu His Ser Gly Asp Leu
Gly Phe Val Asp Asp 490 495 500 aag ggt tat gtt tca ctg acg gga cga
tcc aag gag atc atc att acc 1711 Lys Gly Tyr Val Ser Leu Thr Gly
Arg Ser Lys Glu Ile Ile Ile Thr 505 510 515 gcc ggc ggc gag aac ata
ccg cca gtg cac atc gag aac acg atc aag 1759 Ala Gly Gly Glu Asn
Ile Pro Pro Val His Ile Glu Asn Thr Ile Lys 520 525 530 aag gag ctg
gat gcc att tcc aat gcc ttt ttg gtg ggc gag cag cgc 1807 Lys Glu
Leu Asp Ala Ile Ser Asn Ala Phe Leu Val Gly Glu Gln Arg 535 540 545
aaa tat ctc act gtt ctg atc acc cta aag acc gaa gtg gac aag gat
1855 Lys Tyr Leu Thr Val Leu Ile Thr Leu Lys Thr Glu Val Asp Lys
Asp 550 555 560 565 tcc ggt gag ccg ctg gac gag ctt agc cac gag tcc
tcc gtg tgg gtg 1903 Ser Gly Glu Pro Leu Asp Glu Leu Ser His Glu
Ser Ser Val Trp Val 570 575 580 aaa tcg ctg gga gtg gag cac aag acc
gta tcg gat atc ctg gcc gca 1951 Lys Ser Leu Gly Val Glu His Lys
Thr Val Ser Asp Ile Leu Ala Ala 585 590 595 ggt ccc tgc ccc aag gtg
tgg aag tcc atc gag gat gcc att aag cgg 1999 Gly Pro Cys Pro Lys
Val Trp Lys Ser Ile Glu Asp Ala Ile Lys Arg 600 605 610 gcc aac aag
cag tcc att tcc aat gcc caa aag gtt cag aag ttc acc 2047 Ala Asn
Lys Gln Ser Ile Ser Asn Ala Gln Lys Val Gln Lys Phe Thr 615 620 625
att ctg ccg cac gac ttc tcc att ccc acc ggc gaa ctt gga ccc acc
2095 Ile Leu Pro His Asp Phe Ser Ile Pro Thr Gly Glu Leu Gly Pro
Thr 630 635 640 645 cac cct aaa ggt taagcgcaac gttgtgtcca
agatgtatgc cgatgagatc 2147 His Pro Lys Gly gagaaactat atgcctagat
ttctcactgc aagatcgaaa ccgatgatag ccgcggaact 2207 tgagctttaa
tgtgaatttg aatttaacgg acttccaagc caattgagtg ccacttttaa 2267
tttgatttag gctgatgtta actgttggat attaaactaa gaacaactat ggccctatgc
2327 ctaggtagac acgagcttgc caacgattag gtccagagat catttaatta
gtaactaagt 2387 tttatttttt atatactatt tggttgtacc aactgaacaa
acgaaaattg tttattgtct 2447 gaagagcaac aataaatttg taattagatt
aactaccaaa aaaaaaaaaa aaaaa 2502 2 649 PRT Drosophila melanogaster
2 Met Ser Thr Ile Asp Ala Leu Tyr Asn Arg Pro Gly Pro Asn Arg Leu 1
5 10 15 Arg Gln Ala Asp Ala Tyr Arg Thr Thr Asn Arg Gln Asp Ala Val
Lys 20 25 30 Ile Arg Met Ala Lys Asp Gly Ile Gly Ala Glu Glu Pro
Ile Ser Val 35 40 45 Pro Gly Leu Leu Lys Arg Thr Val Asn Asn Tyr
Gly Asp Tyr Pro Ala 50 55 60 Leu Arg Thr Lys Asn Gly Lys Asn Gly
Tyr His Thr Val Thr Tyr Lys 65 70 75 80 Gln Tyr Glu Gln Lys Val His
Gln Val Ala Lys Ala Phe Ile Lys Leu 85 90 95 Gly Leu Glu Glu His
His Ser Val Gly Val Leu Ala Phe Asn Cys Ala 100 105 110 Glu Trp Phe
Tyr Ser Ala Met Gly Ala Ile His Ala Arg Gly Ile Ile 115 120 125 Ala
Gly Ile Tyr Thr Thr Asn Ser Ala Asp Ala Val Gln His Val Leu 130 135
140 Glu Ser Ser His Ala Gln Ile Val Val Val Asp Asp Ala Lys Gln Met
145 150 155 160 Asp Lys Ile His Ala Ile Arg Asp Lys Leu Pro Lys Leu
Lys Ala Ala 165 170 175 Ile Gln Ile Gln Glu Pro Tyr Ser Pro Tyr Leu
Lys Lys Glu Asp Gly 180 185 190 Tyr Tyr Arg Trp Ser Glu Ile Glu Ser
Met Asn Val Ser Asp Val Glu 195 200 205 Asp Gln Tyr Met Thr Arg Leu
Glu Asn Val Ala Ile Asn Glu Cys Cys 210 215 220 Cys Leu Val Tyr Thr
Ser Gly Thr Val Gly Met Pro Lys Gly Val Met 225 230 235 240 Leu Ser
His Asp Asn Ile Thr Phe Asp Val Arg Gly Ile Val Lys Ala 245 250 255
Met Asp Arg Val Val Val Gly Ala Glu Ser Ile Val Ser Tyr Leu Pro 260
265 270 Leu Ser His Val Ala Ala Gln Thr Val Asp Ile Tyr Thr Cys Ala
Phe 275 280 285 Val Ala Gly Cys Ile Trp Phe Ala Asp Lys Asp Ala Leu
Lys Gly Thr 290 295 300 Leu Val Lys Ser Leu Gln Asp Ala Arg Pro Thr
Arg Phe Met Gly Val 305 310 315 320 Pro Arg Val Tyr Glu Lys Phe Gln
Glu Arg Met Val Ala Val Ala Ser 325 330 335 Ser Ser Gly Ser Leu Lys
Lys Met Leu Ala Ser Trp Ala Lys Gly Ile 340 345 350 Thr Leu Lys His
Tyr Met Val Ser Gln Gly Lys Ser Ser Gly Gly Phe 355 360 365 Arg Tyr
Lys Ile Ala Lys Ser Leu Ile Met Ser Lys Val Lys Gln Ala 370 375 380
Leu Gly Phe Asp Arg Val Leu Thr Leu Ala Ser Ala Ala Ala Pro Met 385
390 395 400 Ser Pro Glu Thr Lys Lys Tyr Phe Leu Ser Leu Asp Leu Lys
Ile Val 405 410 415 Asp Ala Phe Gly Met Ser Glu Thr Ala Gly Cys His
Thr Ile Cys Leu 420 425 430 Pro Asp Ser Val Gly Leu Asn Thr Ile Gly
Lys Thr Leu Pro Gly Cys 435 440 445 Glu Ser Lys Phe Ile Asn Lys Asp
Ala Asn Gly His Gly Glu Leu Cys 450 455 460 Ile Arg Gly Arg His Val
Phe Met Gly Tyr Ile Asp Asn Lys Glu Lys 465 470 475 480 Thr Glu Glu
Ser Leu Asp Asp Asp Cys Trp Leu His Ser Gly Asp Leu 485 490 495 Gly
Phe Val Asp Asp Lys Gly Tyr Val Ser Leu Thr Gly Arg Ser Lys 500 505
510 Glu Ile Ile Ile Thr Ala Gly Gly Glu Asn Ile Pro Pro Val His Ile
515 520 525 Glu Asn Thr Ile Lys Lys Glu Leu Asp Ala Ile Ser Asn Ala
Phe Leu 530 535 540 Val Gly Glu Gln Arg Lys Tyr Leu Thr Val Leu Ile
Thr Leu Lys Thr 545 550 555 560 Glu Val Asp Lys Asp Ser Gly Glu Pro
Leu Asp Glu Leu Ser His Glu 565 570 575 Ser Ser Val Trp Val Lys Ser
Leu Gly Val Glu His Lys Thr Val Ser 580 585 590 Asp Ile Leu Ala Ala
Gly Pro Cys Pro Lys Val Trp Lys Ser Ile Glu 595 600 605 Asp Ala Ile
Lys Arg Ala Asn Lys Gln Ser Ile Ser Asn Ala Gln Lys 610 615 620 Val
Gln Lys Phe Thr Ile Leu Pro His Asp Phe Ser Ile Pro Thr Gly 625 630
635 640 Glu Leu Gly Pro Thr His Pro Lys Gly 645 3 634 PRT Homo
sapiens 3 Arg Leu Arg Ile Asp Pro Ser Cys Pro Gln Leu Pro Tyr Thr
Val His 1 5 10 15 Arg Met Phe Tyr Glu Ala Leu Asp Lys Tyr Gly Asp
Leu Ile Ala Leu 20 25 30 Gly Phe Lys Arg Gln Asp Lys Trp Glu His
Ile Ser Tyr Ser Gln Tyr 35 40 45 Tyr Leu Leu Ala Arg Arg Ala Ala
Lys Gly Phe Leu Lys Leu Gly Leu 50 55 60 Lys Gln Ala His Ser Val
Ala Ile Leu Gly Phe Asn Ser Pro Glu Trp 65 70 75 80 Phe Phe Ser Ala
Val Gly Thr Val Phe Ala Gly Gly Ile Val Thr Gly 85 90 95 Ile Tyr
Thr Thr Ser Ser Pro Glu Ala Cys Gln Tyr Ile Ala Tyr Asp 100 105 110
Cys Cys Ala Asn Val Ile Met Val Asp Thr Gln Lys Gln Leu Glu Lys 115
120 125 Ile Leu Lys Ile Trp Lys Gln Leu Pro His Leu Lys Ala Val Val
Ile 130 135 140 Tyr Lys Glu Pro Pro Pro Asn Lys Met Ala Asn Val Tyr
Thr Met Glu 145 150 155 160 Glu Phe Met Glu Leu Gly Asn Glu Val Pro
Glu Glu Ala Leu Asp Ala 165 170 175 Ile Ile Asp Thr Gln Gln Pro Asn
Gln Cys Cys Val Leu Val Tyr Thr 180 185 190 Ser Gly Thr Thr Gly Asn
Pro Lys Gly Val Met Leu Ser Gln Asp Asn 195 200 205 Ile Thr Trp Thr
Ala Arg Tyr Gly Ser Gln Ala Gly Asp Ile Arg Pro 210 215 220 Ala Glu
Val Gln Gln Glu Val Val Val Ser Tyr Leu Pro Leu Ser His 225 230 235
240 Ile Ala Ala Gln Ile Tyr Asp Leu Trp Thr Gly Ile Gln Trp Gly Ala
245 250 255 Gln Val Cys Phe Ala Glu Pro Asp Ala Leu Lys Gly Ser Leu
Val Asn 260 265 270 Thr Leu Arg Glu Val Glu Pro Thr Ser His Met Gly
Val Pro Arg Val 275 280 285 Trp Glu Lys Ile Met Glu Arg Ile Gln Glu
Val Ala Ala Gln Ser Gly 290 295 300 Phe Ile Arg Arg Lys Met Leu Leu
Trp Ala Met Ser Val Thr Leu Glu 305 310 315 320 Gln Asn Leu Thr Cys
Pro Gly Ser Asp Leu Lys Pro Phe Thr Thr Arg 325 330 335 Leu Ala Asp
Tyr Leu Val Leu Ala Lys Val Arg Gln Ala Leu Gly Phe 340 345 350 Ala
Lys Cys Gln Lys Asn Phe Tyr Gly Ala Ala Pro Met Met Ala Glu 355 360
365 Thr Gln His Phe Phe Leu Gly Leu Asn Ile Arg Leu Tyr Ala Gly Tyr
370 375 380 Gly Leu Ser Glu Thr Ser Gly Pro His Phe Met Ser Ser Pro
Tyr Asn 385 390 395 400 Tyr Arg Leu Tyr Ser Ser Gly Lys Leu Val Pro
Gly Cys Arg Val Lys 405 410 415 Leu Val Asn Gln Asp Ala Glu Gly Ile
Gly Glu Ile Cys Leu Trp Gly 420 425 430 Arg Thr Ile Phe Met Gly Tyr
Leu Asn Met Glu Asp Lys Thr Cys Glu 435 440 445 Ala Ile Asp Glu Glu
Gly Trp Leu His Thr Gly Asp Ala Gly Arg Leu 450 455 460 Asp Ala Asp
Gly Phe Leu Tyr Ile Thr Gly Arg Leu Lys Glu Leu Ile 465 470 475 480
Ile Thr Ala Gly Gly Glu Asn Val Pro Pro Val Pro Ile Glu Glu Ala 485
490 495 Val Lys Met Glu Leu Pro Ile Ile Ser Asn Ala Met Leu Ile Gly
Asp 500 505 510 Gln Arg Lys Phe Leu Ser Met Leu Leu Thr Leu Lys Cys
Thr Leu Asp 515 520 525 Pro Asp Thr Ser Asp Gln Thr Asp Asn Leu Thr
Glu Gln Ala Val Glu 530 535 540 Phe Cys Gln Arg Val Gly Ser Arg Ala
Thr Thr Val Ser Glu Ile Ile 545 550 555 560 Glu Lys Lys Asp Glu Ala
Val Tyr Gln Ala Ile Glu Glu Gly Ile Arg 565 570 575 Arg Val Asn Met
Asn Ala Ala Ala Arg Pro Tyr His Ile Gln Lys Trp 580 585 590 Ala Ile
Leu Glu Arg Asp Phe Ser Ile Ser Gly Gly Glu Leu Gly Pro 595 600 605
Thr Met Lys Leu Lys Arg Leu Thr Val Leu Glu Lys Tyr Lys Gly Ile 610
615 620 Ile Asp Ser Phe Tyr Gln Glu Gln Lys Met 625 630 4 620 PRT
Rattus norvegicus 4 Met Leu Pro Val Leu Tyr
Thr Gly Leu Ala Gly Leu Leu Leu Leu Pro 1 5 10 15 Leu Leu Leu Thr
Cys Cys Cys Pro Tyr Leu Leu Gln Asp Val Arg Phe 20 25 30 Phe Leu
Gln Leu Ala Asn Met Ala Arg Gln Val Arg Ser Tyr Arg Gln 35 40 45
Arg Arg Pro Val Arg Thr Ile Leu His Val Phe Leu Glu Gln Ala Arg 50
55 60 Lys Thr Pro His Lys Pro Phe Leu Leu Phe Arg Asp Glu Thr Leu
Thr 65 70 75 80 Tyr Ala Gln Val Asp Arg Arg Ser Asn Gln Val Ala Arg
Ala Leu His 85 90 95 Asp His Leu Gly Leu Arg Gln Gly Asp Cys Val
Ala Leu Phe Met Gly 100 105 110 Asn Glu Pro Ala Tyr Val Trp Leu Trp
Leu Gly Leu Leu Lys Leu Gly 115 120 125 Cys Pro Met Ala Cys Leu Asn
Tyr Asn Ile Arg Ala Lys Ser Leu Leu 130 135 140 His Cys Phe Gln Cys
Cys Gly Ala Lys Val Leu Leu Ala Ser Pro Glu 145 150 155 160 Leu His
Glu Ala Val Glu Glu Val Leu Pro Thr Leu Lys Lys Glu Gly 165 170 175
Val Ser Val Phe Tyr Val Ser Arg Thr Ser Asn Thr Asn Gly Val Asp 180
185 190 Thr Val Leu Asp Lys Val Asp Gly Val Ser Ala Asp Pro Ile Pro
Glu 195 200 205 Ser Trp Arg Ser Glu Val Thr Phe Thr Thr Pro Ala Val
Tyr Ile Tyr 210 215 220 Thr Ser Gly Thr Thr Gly Leu Pro Lys Ala Ala
Thr Ile Asn His His 225 230 235 240 Arg Leu Trp Tyr Gly Thr Ser Leu
Ala Leu Arg Ser Gly Ile Lys Ala 245 250 255 His Asp Val Ile Tyr Thr
Thr Met Pro Leu Tyr His Ser Ala Ala Leu 260 265 270 Met Ile Gly Leu
His Gly Cys Ile Val Val Gly Ala Thr Phe Ala Leu 275 280 285 Arg Ser
Lys Phe Ser Ala Ser Gln Phe Trp Asp Asp Cys Arg Lys Tyr 290 295 300
Asn Ala Thr Val Ile Gln Tyr Ile Gly Glu Leu Leu Arg Tyr Leu Cys 305
310 315 320 Asn Thr Pro Gln Lys Pro Asn Asp Arg Asp His Lys Val Lys
Ile Ala 325 330 335 Leu Gly Asn Gly Leu Arg Gly Asp Val Trp Arg Glu
Phe Ile Lys Arg 340 345 350 Phe Gly Asp Ile His Ile Tyr Glu Phe Tyr
Ala Ser Thr Glu Gly Asn 355 360 365 Ile Gly Phe Met Asn Tyr Pro Arg
Lys Ile Gly Ala Val Gly Arg Glu 370 375 380 Asn Tyr Leu Gln Lys Lys
Val Val Arg His Glu Leu Ile Lys Tyr Asp 385 390 395 400 Val Glu Lys
Asp Glu Pro Val Arg Asp Ala Asn Gly Tyr Cys Ile Lys 405 410 415 Val
Pro Lys Gly Glu Val Gly Leu Leu Ile Cys Lys Ile Thr Glu Leu 420 425
430 Thr Pro Phe Phe Gly Tyr Ala Gly Gly Lys Thr Gln Thr Glu Lys Lys
435 440 445 Lys Leu Arg Asp Val Phe Lys Lys Gly Asp Val Tyr Phe Asn
Ser Gly 450 455 460 Asp Leu Leu Met Ile Asp Arg Glu Asn Phe Ile Tyr
Phe His Asp Arg 465 470 475 480 Val Gly Asp Thr Phe Arg Trp Lys Gly
Glu Asn Val Ala Thr Thr Glu 485 490 495 Val Ala Asp Ile Val Gly Leu
Val Asp Phe Val Glu Glu Val Asn Val 500 505 510 Tyr Gly Val Pro Val
Pro Gly His Glu Gly Arg Ile Gly Met Ala Ser 515 520 525 Ile Lys Met
Lys Glu Asn Tyr Glu Phe Asn Gly Lys Lys Leu Phe Gln 530 535 540 His
Ile Ser Glu Tyr Leu Pro Ser Tyr Ser Arg Pro Arg Phe Leu Arg 545 550
555 560 Ile Gln Asp Thr Ile Glu Ile Thr Gly Thr Phe Lys His Arg Lys
Val 565 570 575 Thr Leu Met Glu Glu Gly Phe Asn Pro Ser Val Ile Lys
Asp Thr Leu 580 585 590 Tyr Phe Met Asp Asp Thr Glu Lys Thr Tyr Val
Pro Met Thr Glu Asp 595 600 605 Ile Tyr Asn Ala Ile Ile Asp Lys Thr
Leu Lys Leu 610 615 620
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