U.S. patent application number 10/871974 was filed with the patent office on 2005-02-10 for human scad family molecules.
This patent application is currently assigned to INCYTE CORPORATION. Invention is credited to Baughn, Mariah R., Corley, Neil C., Gorgone, Gina A., Guegler, Karl J., Lal, Preeti, Yue, Henry.
Application Number | 20050032098 10/871974 |
Document ID | / |
Family ID | 22326373 |
Filed Date | 2005-02-10 |
United States Patent
Application |
20050032098 |
Kind Code |
A1 |
Lal, Preeti ; et
al. |
February 10, 2005 |
Human SCAD family molecules
Abstract
The invention provides human SCAD family molecules (HSFM) and
polynucleotides which identify and encode HSFM. The invention also
provides expression vectors, host cells, antibodies, agonists, and
antagonists. The invention also provides methods for diagnosing,
treating, or preventing disorders associated with expression of
HSFM.
Inventors: |
Lal, Preeti; (Santa Clara,
CA) ; Guegler, Karl J.; (Menlo Park, CA) ;
Gorgone, Gina A.; (Boulder Creek, CA) ; Corley, Neil
C.; (Mountain View, CA) ; Baughn, Mariah R.;
(San Leandro, CA) ; Yue, Henry; (Sunnyvale,
CA) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
INCYTE CORPORATION
|
Family ID: |
22326373 |
Appl. No.: |
10/871974 |
Filed: |
June 21, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10871974 |
Jun 21, 2004 |
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09470816 |
Dec 23, 1999 |
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09470816 |
Dec 23, 1999 |
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09109205 |
Jun 30, 1998 |
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6057140 |
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Current U.S.
Class: |
435/6.14 ;
435/189; 435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/0006
20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/189; 435/320.1; 435/325; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/02 |
Claims
What is claimed is:
1. A substantially purified polypeptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1. SEQ ID
NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:2.
2. A substantially purified variant having at least 90% amino acid
identity to the amino acid sequence of claim 1.
3. An isolated and purified polynucleotide encoding the polypeptide
of claim 1.
4. An isolated and purified polynucleotide variant having at least
90% polynucleotide sequence identity to the polynucleotide of claim
3.
5. An isolated and purified polynucleotide which hybridizes under
stringent conditions to the polynucleotide of claim 3.
6. An isolated and purified polynucleotide having a sequence which
is complementary to the polynucleotide sequence of claim 3.
7. An isolated and purified polynucleotide comprising a
polynucleotide sequence selected from the group consisting of SEQ
ID NO:3, SEQ ID NO:4, a fragment of SEQ ID NO:3, and a fragment of
SEQ ID NO:4.
8. An isolated and purified polynucleotide variant having at least
90% polynucleotide sequence identity to the polynucleotide of claim
7.
9. An isolated and purified polynucleotide having a sequence which
is complementary to the polynucleotide of claim 7.
10. An expression vector comprising at least a fragment of the
polynucleotide of claim 3.
11. A host cell comprising the expression vector of claim 10.
12. A method for producing a polypeptide comprising the amino acid
sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:2, the
method comprising the steps of: a) culturing the host cell of claim
11 under conditions suitable for the expression of the polypeptide;
and b) recovering the polypeptide from the host cell culture.
13. A pharmaceutical composition comprising the polypeptide of
claim 1 in conjunction with a suitable pharmaceutical carrier.
14. A purified antibody which specifically binds to the polypeptide
of claim 1.
15. A purified agonist of the polypeptide of claim 1.
16. A purified antagonist of the polypeptide of claim 1.
17. A method for treating or preventing a cell proliferative
disorder, the method comprising administering to a subject in need
of such treatment an effective amount of the pharmaceutical
composition of claim 13.
18. A method for treating or preventing an inflammatory disorder,
the method comprising administering to a subject in need of such
treatment an effective amount of the pharmaceutical composition of
claim 13.
19. A method for treating or preventing a fatty acid and steroid
metabolic disorder, the method comprising administering to a
subject in need of such treatment an effective amount of the
pharmaceutical composition of claim 13.
20. A method for detecting a polynucleotide encoding the
polypeptide comprising the amino acid sequence selected from the
group consisting of SEQ ID NO:1, SEQ ID NO:2, a fragment of SEQ ID
NO:1, and a fragment of SEQ ID NO:2 in a biological sample, the
method comprising the steps of: (a) hybridizing the polynucleotide
of claim 6 to at least one of the nucleic acids in the biological
sample, thereby forming a hybridization complex; and (b) detecting
the hybridization complex, wherein the presence of the
hybridization complex correlates with the presence of the
polynucleotide encoding the polypeptide in the biological
sample.
21. The method of claim 20 further comprising amplifying the
polynucleotide prior to the hybridizing step.
Description
FIELD OF THE INVENTION
[0001] This invention relates to nucleic acid and amino acid
sequences of human SCAD family molecules and to the use of these
sequences in the diagnosis, treatment, and prevention of disorders
associated with cell proliferation, inflammation, and fatty acid
and steroid metabolism.
BACKGROUND OF THE INVENTION
[0002] The short-chain alcohol dehydrogenases (SCADs) are a diverse
family of oxidoreductase enzymes. SCAD family members are involved
in all aspects of cell biochemistry and physiology, including
metabolism of sugar, synthesis or degradation of fatty acids, and
synthesis or degradation of glucocorticoids, estrogens, androgens,
and prostaglandins E.sub.2 and F.sub.2.alpha.. SCADs are found in
bacteria, plants, invertebrates, and vertebrates. Alignment of the
different family members reveals large homologous regions and
clustered similarities indicating sites of structural and
functional importance. Some of these sites are associated with a
type of coenzyme-binding domain, but similarity between family
members extends beyond this domain. Family members typically show
only about 15% to 30% identity between enzyme pairs. Over one third
of the conserved residues are glycine residues, showing the
importance of conformational and spatial restrictions. (Baker, M.
E. (1995) Biochem. J. 309:1029-1030; and Jomvall, H. et al. (1995)
Biochemistry 34:6003-6013.) SCAD family members show different
subcellular distributions. For example, 2,4-dienoyl-CoA reductase
is located in the mitochondria, whereas retinol dehydrogenase is
located in microsomes.
[0003] The SCAD family can be divided into two groups based on the
arrangement of two conserved structural motifs. The first group
contains a highly conserved pentapeptide, containing a tyrosine and
a lysine, separated by any three amino acid residues, at about
residue 150 in a 250-residue dehydrogenase. The tyrosine and lysine
residues, which are absolutely conserved within this group, are
likely to be important in catalysis. Support for the importance of
these two residues comes from mutagenesis studies with Drosophila
alcohol dehydrogenase, human 15-hydroxyprostaglandin dehydrogenase,
and human 11.beta.-hydroxysteroid and 17-.beta.-hydroxysteroid
dehydrogenases. (Baker, supra.) The AMP-binding domain at the
N-terminus, which consists of a hydrophobic pocket containing three
glycine residues in a seven amino acid sequence, is also highly
conserved in this group. (Baker, supra.) The second group lacks
either the tyrosine or the lysine in the pentapeptide motif. For
example, the tyrosine residue is replaced by a methionine in E.
coli enoyl-acyl-carrier protein (EnvM), by serine in rat and human
2,4-dienoyl-CoA reductases, and by valine in S. cerevisiae
sporulation specific protein (SPX19). Some members of this group
also have differences in the AMP-binding domain, including an
insertion of two residues and poor conservation of the second and
third glycine residues. These changes do not seem to affect the
enoyl-CoA reductase activity of the proteins, though in the case of
EnvM NAD.sup.+ and substrate must bind simultaneously. (Baker,
supra.)
[0004] The members of the SCAD family share a common function,
utilizing NAD.sup.+ or NADP as a cofactor in oxidation-reduction
reactions, but differ in their substrate specificity. For example,
17-.beta.-hydroxysteroid dehydrogenase interconverts estrone and
estradiol, and androstenedione and testosterone. 2,4-dienoyl-CoA
reductase participates in the metabolism of unsaturated fatty
acids, and 15-hydroxyprostaglandin dehydrogenase is the main enzyme
in prostaglandin degradation. Retinol dehydrogenase catalyzes the
primary rate limiting step in retinoic acid synthesis, and
11-cis-retinol dehydrogenase catalyzes the final step in the
biosynthesis of 11-cis-retinaldehyde, the universal chromophore of
visual pigments.
[0005] SCAD involvement in fatty acid and steroid metabolism
implicates members of the SCAD family in a variety of disorders.
Steroid dehydrogenases, such as the hydroxysteroid dehydrogenases,
are involved in hypertension, fertility, and cancer. (Duax, W. L.
and Ghosh, D. (1997) Steroids 62:95-100.) Reduction in
2,4-dienoyl-CoA reductase activity has been associated with
hyperlysinemia and hypocamitinemia. (Roe, C. R. et al. (1990) J.
Clin. Invest. 85:1703-1707.) Retinoic acid, a regulator of
differentiation and apoptosis, has been shown to down-regulate
genes involved in cell proliferation and inflammation. (Chai, X. et
al. (1995) J. Biol. Chem. 270:3900-3904.) The discovery of new
human SCAD family molecules and the polynucleotides encoding them
satisfies a need in the art by providing new compositions which are
useful in the diagnosis, treatment, and prevention of disorders
associated with proliferation, inflammation, and fatty acid and
steroid metabolism.
SUMMARY OF THE INVENTION
[0006] The invention features substantially purified polypeptides,
human SCAD family molecules, referred to collectively as "HSFM" and
individually as "HSFM-1" and "HSFM-2." In one aspect, the invention
provides a substantially purified polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1,
SEQ ID NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID
NO:2.
[0007] The invention further provides a substantially purified
variant having at least 90% amino acid identity to the amino acid
sequences of SEQ ID NO:1 or SEQ ID NO:2, or to a fragment of either
of these sequences. The invention also provides an isolated and
purified polynucleotide encoding the polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO:1, SEQ ID NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ
ID NO:2. The invention also includes an isolated and purified
polynucleotide variant having at least 90% polynucleotide seqeunce
identity to the polynucleotide encoding the polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID
NO:1, SEQ ID NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ
ID NO:2.
[0008] Additionally, the invention provides an isolated and
purified polynucleotide which hybridizes under stringent conditions
to the polynucleotide encoding the polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1,
SEQ ID NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID
NO:2, as well as an isolated and purified polynucleotide having a
sequence which is complementary to the polynucleotide encoding the
polypeptide comprising the amino acid sequence selected from the
group consisting of SEQ ID NO:1, SEQ ID NO:2, a fragment of SEQ ID
NO:1, and a fragment of SEQ ID NO:2.
[0009] The invention also provides an isolated and purified
polynucleotide comprising a polynucleotide sequence selected from
the group consisting of SEQ ID NO:3, SEQ ID NO:4, a fragment of SEQ
ID NO:3, and a fragment of SEQ ID NO:4. The invention further
provides an isolated and purified polynucleotide variant having at
least 90% polynucleotide sequence identity to the polynucleotide
sequence comprising a polynucleotide sequence selected from the
group consisting of SEQ ID NO:3, SEQ ID NO:4, a fragment of SEQ ID
NO:3, and a fragment of SEQ ID NO:4, as well as an isolated and
purified polynucleotide having a sequence which is complementary to
the polynucleotide comprising a polynucleotide sequence selected
from the group consisting of SEQ ID NO:3, SEQ ID NO:4, a fragment
of SEQ ID NO:3, and a fragment of SEQ ID NO:4.
[0010] The invention further provides an expression vector
containing at least a fragment of the polynucleotide encoding the
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:1, SEQ ID NO:2, a fragment of SEQ ID
NO:1, and a fragment of SEQ ID NO:2. In another aspect, the
expression vector is contained within a host cell.
[0011] The invention also provides a method for producing a
polypeptide comprising the amino acid sequence selected from the
group consisting of SEQ ID NO:1, SEQ ID NO:2, a fragment of SEQ ID
NO:1, and a fragment of SEQ ID NO:2, the method comprising the
steps of: (a) culturing the host cell containing an expression
vector containing at least a fragment of a polynucleotide encoding
the polypeptide under conditions suitable for the expression of the
polypeptide; and (b) recovering the polypeptide from the host cell
culture.
[0012] The invention also provides a pharmaceutical composition
comprising a substantially purified polypeptide having the amino
acid sequence selected from the group consisting of SEQ ID NO:1,
SEQ ID NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID
NO:2 in conjunction with a suitable pharmaceutical carrier.
[0013] The invention further includes a purified antibody which
binds to a polypeptide comprising the amino acid sequence selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:2, a fragment
of SEQ ID NO:1, and a fragment of SEQ ID NO:2, as well as a
purified agonist and a purified antagonist to the polypeptide. The
invention also provides a method for treating or preventing a cell
proliferative disorder, the method comprising administering to a
subject in need of such treatment an effective amount of a
pharmaceutical composition comprising a substantially purified
polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:2, a fragment of SEQ ID NO:1,
and a fragment of SEQ ID NO:2.
[0014] The invention also provides a method for treating or
preventing an inflammatory disorder, the method comprising
administering to a subject in need of such treatment an effective
amount of a pharmaceutical composition comprising a substantially
purified polypeptide having an amino acid sequence selected from
the group consisting of SEQ ID NO:1, SEQ ID NO:2, a fragment of SEQ
ID NO:1, and a fragment of SEQ ID NO:2.
[0015] The invention also provides a method for treating or
preventing a fatty acid and steroid metabolic disorder, the method
comprising administering to a subject in need of such treatment an
effective amount of a pharmaceutical composition comprising a
substantially purified polypeptide having an amino acid sequence
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, a
fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:2.
[0016] The invention also provides a method for detecting a
polynucleotide encoding the polypeptide comprising the amino acid
sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:2 in a
biological sample containing nucleic acids, the method comprising
the steps of: (a) hybridizing the complement of the polynucleotide
sequence encoding the polypeptide comprising the amino acid
sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:2 to
at least one of the nucleic acids of the biological sample, thereby
forming a hybridization complex; and (b) detecting the
hybridization complex, wherein the presence of the hybridization
complex correlates with the presence of a polynucleotide encoding
the polypeptide in the biological sample. In one aspect, the method
further comprises amplifying the polynucleotide prior to the
hybridizing step.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIGS. 1A, 1B, 1C, and 1D show the amino acid sequence (SEQ
ID NO:1) and nucleic acid sequence (SEQ ID NO:3) of HSFM-1. The
alignments were produced using MacDNASIS PRO.TM. software (Hitachi
Software Engineering Co. Ltd., San Bruno, Calif.).
[0018] FIGS. 2A, 2B, 2C, 2D, and 2E show the amino acid sequence
(SEQ ID NO:2) and nucleic acid sequence (SEQ ID NO:4) of
HSFM-2.
[0019] FIGS. 3A and 3B show the amino acid sequence alignments
among HSFM-I (1511003; SEQ ID NO:1), human 11-cis-retinol
dehydrogenase (GI 1616654; SEQ ID NO:17) and rat retinol
dehydrogenase (GI 841197; SEQ ID NO:18)
[0020] FIGS. 4A and 4B show the amino acid sequence alignments
between HSFM-2 (1810320; SEQ ID NO:2) and human 2,4-dienoyl-CoA
reductase (GI 1575000; SEQ ID NO:19). Sequence alignments were
produced using the multisequence alignment program of LASERGENE.TM.
software (DNASTAR Inc, Madison Wis.).
DESCRIPTION OF THE INVENTION
[0021] Before the present proteins, nucleotide sequences, and
methods are described, it is understood that this invention is not
limited to the particular methodology, protocols, cell lines,
vectors, and reagents described, as these may vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
limit the scope of the present invention which will be limited only
by the appended claims.
[0022] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, a reference to "a host cell" includes a plurality of such
host cells, and a reference to "an antibody" is a reference to one
or more antibodies and equivalents thereof known to those skilled
in the art, and so forth.
[0023] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods, devices, and materials are now
described. All publications mentioned herein are cited for the
purpose of describing and disclosing the cell lines, vectors, and
methodologies which are reported in the publications and which
might be used in connection with the invention. Nothing herein is
to be construed as an admission that the invention is not entitled
to antedate such disclosure by virtue of prior invention.
[0024] Definitions
[0025] "HSFM," as used herein, refers to the amino acid sequences,
or variant thereof, of substantially purified HSFM obtained from
any species, particularly a mammalian species, including bovine,
ovine, porcine, murine, equine, and preferably the human species,
from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
[0026] The term "agonist," as used herein, refers to a molecule
which, when bound to HSFM, increases or prolongs the duration of
the effect of HSFM. Agonists may include proteins, nucleic acids,
carbohydrates, or any other molecules which bind to and modulate
the effect of HSFM.
[0027] An "allelic variant," as this term is used herein, is an
alternative form of the gene encoding HSFM. Allelic variants may
result from at least one mutation in the nucleic acid sequence and
may result in altered mRNAs or in polypeptides whose structure or
function may or may not be altered. Any given natural or
recombinant gene may have none, one, or many allelic forms. Common
mutational changes which give rise to allelic variants are
generally ascribed to natural deletions, additions, or
substitutions of nucleotides. Each of these types of changes may
occur alone, or in combination with the others, one or more times
in a given sequence.
[0028] "Altered" nucleic acid sequences encoding HSFM, as described
herein, include those sequences with deletions, insertions, or
substitutions of different nucleotides, resulting in a
polynucleotide the same as HSFM or a polypeptide with at least one
functional characteristic of HSFM. Included within this definition
are polymorphisms which may or may not be readily detectable using
a particular oligonucleotide probe of the polynucleotide encoding
HSFM, and improper or unexpected hybridization to allelic variants,
with a locus other than the normal chromosomal locus for the
polynucleotide sequence encoding HSFM. The encoded protein may also
be "altered," and may contain deletions, insertions, or
substitutions of amino acid residues which produce a silent change
and result in a functionally equivalent HSFM. Deliberate amino acid
substitutions may be made on the basis of similarity in polarity,
charge, solubility, hydrophobicity, hydrophilicity, and/or the
amphipathic nature of the residues, as long as the biological or
immunological activity of HSFM is retained. For example, negatively
charged amino acids may include aspartic acid and glutamic acid,
positively charged amino acids may include lysine and arginine, and
amino acids with uncharged polar head groups having similar
hydrophilicity values may include leucine, isoleucine, and valine;
glycine and alanine; asparagine and glutamine; serine and
threonine; and phenylalanine and tyrosine.
[0029] The terms "amino acid" or "amino acid sequence," as used
herein, refer to an oligopeptide, peptide, polypeptide, or protein
sequence, or a fragment of any of these, and to naturally occurring
or synthetic molecules. In this context, "fragments," "immunogenic
fragments," or "antigenic fragments" refer to fragments of HSFM
which are preferably at least 5 to about 15 amino acids in length,
most preferably at least 14 amino acids, and which retain some
biological activity or immunological activity of HSFM. Where "amino
acid sequence" is recited herein to refer to an amino acid sequence
of a naturally occurring protein molecule, "amino acid sequence"
and like terms are not meant to limit the amino acid sequence to
the complete native amino acid sequence associated with the recited
protein molecule.
[0030] "Amplification," as used herein, relates to the production
of additional copies of a nucleic acid sequence. Amplification is
generally carried out using polymerase chain reaction (PCR)
technologies well known in the art. (See, e.g., Dieffenbach, C. W.
and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold
Spring Harbor Press, Plainview, N.Y., pp. 1-5.)
[0031] The term "antagonist," as it is used herein, refers to a
molecule which, when bound to HSFM, decreases the amount or the
duration of the effect of the biological or immunological activity
of HSFM. Antagonists may include proteins, nucleic acids,
carbohydrates, antibodies, or any other molecules which decrease
the effect of HSFM.
[0032] As used herein, the term "antibody" refers to intact
molecules as well as to fragments thereof, such as Fab,
F(ab').sub.2, and Fv fragments, which are capable of binding the
epitopic determinant. Antibodies that bind HSFM polypeptides can be
prepared using intact polypeptides or using fragments containing
small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide used to immunize an animal (e.g., a
mouse, a rat, or a rabbit) can be derived from the translation of
RNA, or synthesized chemically, and can be conjugated to a carrier
protein if desired. Commonly used carriers that are chemically
coupled to peptides include bovine serum albumin, thyroglobulin,
and keyhole limpet hemocyanin (KLH). The coupled peptide is then
used to immunize the animal.
[0033] The term "antigenic determinant," as used herein, refers to
that fragment of a molecule (i.e., an epitope) that makes contact
with a particular antibody. When a protein or a fragment of a
protein is used to immunize a host animal, numerous regions of the
protein may induce the production of antibodies which bind
specifically to antigenic determinants (given regions or
three-dimensional structures on the protein). An antigenic
determinant may compete with the intact antigen (i.e., the
immunogen used to elicit the immune response) for binding to an
antibody.
[0034] The term "antisense," as used herein, refers to any
composition containing a nucleic acid sequence which is
complementary to the "sense" strand of a specific nucleic acid
sequence. Antisense molecules may be produced by any method
including synthesis or transcription. Once introduced into a cell,
the complementary nucleotides combine with natural sequences
produced by the cell to form duplexes and to block either
transcription or translation. The designation "negative" can refer
to the antisense strand, and the designation "positive" can refer
to the sense strand.
[0035] As used herein, the term "biologically active," refers to a
protein having structural, regulatory, or biochemical functions of
a naturally occurring molecule. Likewise, "immunologically active"
refers to the capability of the natural, recombinant, or synthetic
HSFM, or of any oligopeptide thereof, to induce a specific immune
response in appropriate animals or cells and to bind with specific
antibodies.
[0036] The terms "complementary" or "complementarity," as used
herein, refer to the natural binding of polynucleotides by base
pairing. For example, the sequence "5' A-G-T 3'" binds to the
complementary sequence "3' T-C-A 5'." Complementarity between two
single-stranded molecules may be "partial," such that only some of
the nucleic acids bind, or it may be "complete," such that total
complementarity exists between the single stranded molecules. The
degree of complementarity between nucleic acid strands has
significant effects on the efficiency and strength of the
hybridization between the nucleic acid strands. This is of
particular importance in amplification reactions, which depend upon
binding between nucleic acids strands, and in the design and use of
peptide nucleic acid (PNA) molecules.
[0037] A "composition comprising a given polynucleotide sequence"
or a "composition comprising a given amino acid sequence," as these
terms are used herein, refer broadly to any composition containing
the given polynucleotide or amino acid sequence. The composition
may comprise a dry formulation or an aqueous solution. Compositions
comprising polynucleotide sequences encoding HSFM or fragments of
HSFM may be employed as hybridization probes. The probes may be
stored in freeze-dried form and may be associated with a
stabilizing agent such as a carbohydrate. In hybridizations, the
probe may be deployed in an aqueous solution containing salts,
e.g., NaCl, detergents, e.g., sodium dodecyl sulfate (SDS), and
other components, e.g., Denhardt's solution, dry milk, salmon sperm
DNA, etc.
[0038] "Consensus sequence," as used herein, refers to a nucleic
acid sequence which has been resequenced to resolve uncalled bases,
extended using XL-PCR.TM. (The Perkin-Elmer Corp., Norwalk, Conn.)
in the 5' and/or the 3' direction, and resequenced, or which has
been assembled from the overlapping sequences of more than one
Incyte Clone using a computer program for fragment assembly, such
as the GELVIEW.TM. Fragment Assembly system (GCG, Madison, Wis.).
Some sequences have been both extended and assembled to produce the
consensus sequence.
[0039] As used herein, the term "correlates with expression of a
polynucleotide" indicates that the detection of the presence of
nucleic acids, the same or related to a nucleic acid sequence
encoding HSFM, by Northern analysis is indicative of the presence
of nucleic acids encoding HSFM in a sample, and thereby correlates
with expression of the transcript from the polynucleotide encoding
HSFM.
[0040] A "deletion," as the term is used herein, refers to a change
in the amino acid or nucleotide sequence that results in the
absence of one or more amino acid residues or nucleotides.
[0041] The term "derivative," as used herein, refers to the
chemical modification of a polypeptide sequence, or a
polynucleotide sequence. Chemical modifications of a polynucleotide
sequence can include, for example, replacement of hydrogen by an
alkyl, acyl, or amino group. A derivative polynucleotide encodes a
polypeptide which retains at least one biological or immunological
function of the natural molecule. A derivative polypeptide is one
modified by glycosylation, pegylation, or any similar process that
retains at least one biological or immunological function of the
polypeptide from which it was derived.
[0042] The term "similarity," as used herein, refers to a degree of
complementarity. There may be partial similarity or complete
similarity. The word "identity" may substitute for the word
"similarity." A partially complementary sequence that at least
partially inhibits an identical sequence from hybridizing to a
target nucleic acid is referred to as "substantially similar." The
inhibition of hybridization of the completely complementary
sequence to the target sequence may be examined using a
hybridization assay (Southern or Northern blot, solution
hybridization, and the like) under conditions of reduced
stringency. A substantially similar sequence or hybridization probe
will compete for and inhibit the binding of a completely similar
(identical) sequence to the target sequence under conditions of
reduced stringency. This is not to say that conditions of reduced
stringency are such that non-specific binding is permitted, as
reduced stringency conditions require that the binding of two
sequences to one another be a specific (i.e., a selective)
interaction. The absence of non-specific binding may be tested by
the use of a second target sequence which lacks even a partial
degree of complementarity (e.g., less than about 30% similarity or
identity). In the absence of non-specific binding, the
substantially similar sequence or probe will not hybridize to the
second non-complementary target sequence.
[0043] The phrases "percent identity" or "% identity" refer to the
percentage of sequence similarity found in a comparison of two or
more amino acid or nucleic acid sequences. Percent identity can be
determined electronically, e.g., by using the MegAligm.TM. program
(DNASTAR, Inc., Madison Wis.). The MegAlign.TM. program can create
alignments between two or more sequences according to different
methods, e.g., the clustal method. (See, e.g., Higgins, D. G. and
P. M. Sharp (1988) Gene 73:237-244.) The clustal algorithm groups
sequences into clusters by examining the distances between all
pairs. The clusters are aligned pairwise and then in groups. The
percentage similarity between two amino acid sequences, e.g.,
sequence A and sequence B, is calculated by dividing the length of
sequence A, minus the number of gap residues in sequence A, minus
the number of gap residues in sequence B, into the sum of the
residue matches between sequence A and sequence B, times one
hundred. Gaps of low or of no similarity between the two amino acid
sequences are not included in determining percentage similarity.
Percent identity between nucleic acid sequences can also be counted
or calculated by other methods known in the art, e.g., the Jotun
Hein method. (See, e.g., Hein, J. (1990) Methods Enzymol.
183:626-645.) Identity between sequences can also be determined by
other methods known in the art, e.g., by varying hybridization
conditions.
[0044] "Human artificial chromosomes" (HACs), as described herein,
are linear microchromosomes which may contain DNA sequences of
about 6 kb to 10 Mb in size, and which contain all of the elements
required for stable mitotic chromosome segregation and maintenance.
(See, e.g., Harrington, J. J. et al. (1997) Nat Genet.
15:345-355.)
[0045] The term "humanized antibody," as used herein, refers to
antibody molecules in which the amino acid sequence in the
non-antigen binding regions has been altered so that the antibody
more closely resembles a human antibody, and still retains its
original binding ability.
[0046] "Hybridization," as the term is used herein, refers to any
process by which a strand of nucleic acid binds with a
complementary strand through base pairing.
[0047] As used herein, the term "hybridization complex" refers to a
complex formed between two nucleic acid sequences by virtue of the
formation of hydrogen bonds between complementary bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot
analysis) or formed between one nucleic acid sequence present in
solution and another nucleic acid sequence immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass
slides, or any other appropriate substrate to which cells or their
nucleic acids have been fixed).
[0048] The words "insertion" or "addition," as used herein, refer
to changes in an amino acid or nucleotide sequence resulting in the
addition of one or more amino acid residues or nucleotides,
respectively, to the sequence found in the naturally occurring
molecule.
[0049] "Immune response" can refer to conditions associated with
inflammation, trauma, immune disorders, or infectious or genetic
disease, etc. These conditions can be characterized by expression
of various factors, e.g., cytokines, chemokines, and other
signaling molecules, which may affect cellular and systemic defense
systems.
[0050] The term "microarray," as used herein, refers to an
arrangement of distinct polynucleotides arrayed on a substrate,
e.g., paper, nylon or any other type of membrane, filter, chip,
glass slide, or any other suitable solid support.
[0051] The terms "element" or "array element" as used herein in a
microarray context; refer to hybridizable polynucleotides arranged
on the surface of a substrate.
[0052] The term "modulate," as it appears herein, refers to a
change in the activity of HSFM. For example, modulation may cause
an increase or a decrease in protein activity, binding
characteristics, or any other biological, functional, or
immunological properties of HSFM.
[0053] The phrases "nucleic acid" or "nucleic acid sequence," as
used herein, refer to a nucleotide, oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer
to DNA or RNA of genomic or synthetic origin which may be
single-stranded or double-stranded and may represent the sense or
the antisense strand, to peptide nucleic acid (PNA), or to any
DNA-like or RNA-like material. In this context, "fragments" refers
to those nucleic acid sequences which, when translated, would
produce polypeptides retaining some functional characteristic,
e.g., antigenicity, or structural domain characteristic, e.g.,
ATP-binding site, of the full-length polypeptide.
[0054] The terms "operably associated" or "operably linked," as
used herein, refer to functionally related nucleic acid sequences.
A promoter is operably associated or operably linked with a coding
sequence if the promoter controls the translation of the encoded
polypeptide. While operably associated or operably linked nucleic
acid sequences can be contiguous and in the same reading frame,
certain genetic elements, e.g. repressor genes, are not
contiguously linked to the sequence encoding the polypeptide but
still bind to operator sequences that control expression of the
polypeptide.
[0055] The term "oligonucleotide," as used herein, refers to a
nucleic acid sequence of at least about 6 nucleotides to 60
nucleotides, preferably about 15 to 30 nucleotides, and most
preferably about 20 to 25 nucleotides, which can be used in PCR
amplification or in a hybridization assay or microarray. As used
herein, the term "oligonucleotide" is substantially equivalent to
the terms "amplimer," "primer," "oligomer," and "probe," as these
terms are commonly defined in the art.
[0056] "Peptide nucleic acid" (PNA), as used herein, refers to an
antisense molecule or anti-gene agent which comprises an
oligonucleotide of at least about 5 nucleotides in length linked to
a peptide backbone of amino acid residues ending in lysine. The
terminal lysine confers solubility to the composition. PNAs
preferentially bind complementary single stranded DNA or RNA and
stop transcript elongation, and may be pegylated to extend their
lifespan in the cell. (See, e.g., Nielsen, P. E. et al. (1993)
Anticancer Drug Des. 8:53-63.)
[0057] The term "sample," as used herein, is used in its broadest
sense. A biological sample suspected of containing nucleic acids
encoding HSFM, or fragments thereof, or HSFM itself, may comprise a
bodily fluid; an extract from a cell, chromosome, organelle, or
membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA,
in solution or bound to a solid support; a tissue; a tissue print;
etc.
[0058] As used herein, the terms "specific binding" or
"specifically binding" refer to that interaction between a protein
or peptide and an agonist, an antibody, or an antagonist. The
interaction is dependent upon the presence of a particular
structure of the protein, e.g., the antigenic determinant or
epitope, recognized by the binding molecule. For example, if an
antibody is specific for epitope "A," the presence of a polypeptide
containing the epitope A, or the presence of free unlabeled A, in a
reaction containing free labeled A and the antibody will reduce the
amount of labeled A that binds to the antibody.
[0059] As used herein, the term "stringent conditions" refers to
conditions which permit hybridization between polynucleotides and
the claimed polynucleotides. Stringent conditions can be defined by
salt concentration, the concentration of organic solvent, e.g.,
formamide, temperature, and other conditions well known in the art.
In particular, stringency can be increased by reducing the
concentration of salt, increasing the concentration of formamide,
or raising the hybridization temperature.
[0060] The term "substantially purified," as used herein, refers to
nucleic acid or amino acid sequences that are removed from their
natural environment and are isolated or separated, and are at least
about 60% free, preferably about 75% free, and most preferably
about 90% free from other components with which they are naturally
associated.
[0061] A "substitution," as used herein, refers to the replacement
of one or more amino acids or nucleotides by different amino acids
or nucleotides, respectively.
[0062] "Transformation," as defined herein, describes a process by
which exogenous DNA enters and changes a recipient cell.
Transformation may occur under natural or artificial conditions
according to various methods well known in the art, and may rely on
any known method for the insertion of foreign nucleic acid
sequences into a prokaryotic or eukaryotic host cell. The method
for transformation is selected based on the type of host cell being
transformed and may include, but is not limited to, viral
infection, electroporation, heat shock, lipofection, and particle
bombardment. The term "transformed" cells includes stably
transformed cells in which the inserted DNA is capable of
replication either as an autonomously replicating plasmid or as
part of the host chromosome, as well as transiently transformed
cells which express the inserted DNA or RNA for limited periods of
time.
[0063] A "variant" of HSFM polypeptides, as used herein, refers to
an amino acid sequence that is altered by one or more amino acid
residues. The variant may have "conservative" changes, wherein a
substituted amino acid has similar structural or chemical
properties (e.g., replacement of leucine with isoleucine).
[0064] More rarely, a variant may have "nonconservative" changes
(e.g., replacement of glycine with tryptophan). Analogous minor
variations may also include amino acid deletions or insertions, or
both. Guidance in determining which amino acid residues may be
substituted, inserted, or deleted without abolishing biological or
immunological activity may be found using computer programs well
known in the art, for example, LASERGENE.TM. software.
[0065] The term "variant," when used in the context of a
polynucleotide sequence, may encompass a polynucleotide sequence
related to HSFM. This definition may also include, for example,
"allelic" (as defined above), "splice," "species," or "polymorphic"
variants. A splice variant may have significant identity to a
reference molecule, but will generally have a greater or lesser
number of polynucleotides due to alternate splicing of exons during
mRNA processing. The corresponding polypeptide may possess
additional functional domains or an absence of domains. Species
variants are polynucleotide sequences that vary from one species to
another. The resulting polypeptides generally will have significant
amino acid identity relative to each other. A polymorphic variant
is a variation in the polynucleotide sequence of a particular gene
between individuals of a given species. Polymorphic variants also
may encompass "single nucleotide polymorphisms" (SNPs) in which the
polynucleotide sequence varies by one base. The presence of SNPs
may be indicative of, for example, a certain population, a disease
state, or a propensity for a disease state.
THE INVENTION
[0066] The invention is based on the discovery of new human SCAD
family molecules (HSFM), the polynucleotides encoding HSFM, and the
use of these compositions for the diagnosis, treatment, or
prevention of disorders associated with cell proliferation,
inflammation, and fatty acid and steroid metabolism.
[0067] Nucleic acids encoding the HSFM-1 of the present invention
were first identified in Incyte Clone 1511003 from the lung cDNA
library (LUNGNOT14) using a computer search, e.g., BLAST, for amino
acid sequence alignments. A consensus sequence, SEQ ID NO:3, was
derived from the following overlapping and/or extended nucleic acid
sequences: Incyte Clones 2921571H1 (SININOT04), 1511003F6,
1511003H1, and 1511003T6 (LUNGNOT14), and 2722958F6
(LUNGTUT10).
[0068] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:1, as shown in
FIGS. 1A, 1B, 1C, and 1D. HSFM-1 is 309 amino acids in length and
has a potential amidation site at residue T130; two potential
casein kinase II phosphorylation sites at residues S21 and S100;
and six potential protein kinase C phosphorylation sites at
residues S7, S8, T80, S100, T130, and T156. BLOCKS identifies
significant sequence identity with short chain alcohol
dehydrogenases from residues E41 through G53, G117 through G127,
and G170 through E207. PRINTS identifies significant sequence
identitiy with short chain alcohol dehydrogenases from residues
G117 through I128, and Y190 through F209. Profilescan identifies a
short-chain alcohol dehydrogenase family signature from residues
D168 through P223. PFAM identifies significant sequence identity
with short chain alcohol dehydrogenases. HSFM-1 contains the
canonical AMP-binding domain and catalytic site of short chain
alcohol dehydrogenases at residues 47-53 and 190-194, respectively.
As shown in FIGS. 3A and 3B, HSFM-1 has chemical and structural
similarity with human 11-cis-retinol dehydrogenase (GI 1616654; SEQ
ID NO:17) and rat retinol dehydrogenase (GI 841197; SEQ ID NO:18).
In particular, HSFM-1 shares 17% identity with human 11-cis-retinol
dehydrogenase and 16% identity with rat retinol dehydrogenase.
HSFM-1, human 11-cis-retinol dehydrogenase, and rat retinol
dehydrogenase also have similar molecular mass (34.1 kDa, 35.0 kDa,
and 35.7 kDa, respectively) and share canonical AMP-binding and
catalytic domains. A region of unique sequence in HSFM-1 from about
amino acid 151 to about amino acid 157 is encoded by a fragment of
SEQ ID NO:3 from about nucleotide 534 to about nucleotide 554.
Northern analysis shows the expression of this sequence in various
libraries, at least 64% of which are proliferative or cancerous and
at least 14% of which involve immune response. Of particular note
is the expression of HSFM-1 in cardiovascular, gastrointestinal,
reproductive, and nervous tissues.
[0069] Nucleic acids encoding the HSFM-2 of the present invention
were first identified in Incyte Clone 1810320 from the prostate
tumor cDNA library (PROSTUT12) using a computer search, e.g.,
BLAST, for amino acid sequence alignments. A consensus sequence,
SEQ ID NO:4, was derived from the following overlapping and/or
extended nucleic acid sequences: Incyte Clones 1515168H1
(PANCTUT01), 1810320H1 (PROSTUT12), 1653184T6 (PROSTUT18),
1750778T6 (LIVRTUT01), 484767.times.17 (HNT2RAT01), and 2466459T6
(THYRNOT08) and shotgun sequence SAFC01552F1.
[0070] In another embodiment, the invention encompasses a
polypeptide comprising the amino acid sequence of SEQ ID NO:2, as
shown in FIGS. 2A, 2B, 2C, 2D, and 2E. HSFM-2 is 292 amino acids in
length and has two potential casein kinase II phosphorylation sites
at residues T132 and S212; four potential protein kinase C
phosphorylation sites at residues T76, S180, S228, and S289; three
potential N-glycosylation sites at residues N143, N162, and N241;
and a C-terminal microbody targeting signal at residues
A.sub.290KL. BLOCKS identifies significant sequence identity with
short chain alcohol dehydrogenase from residues K29 through G41,
G105 through A115, G158 through E195, and N200 through G209. PRINTS
identifies significant sequence identity with short chain alcohol
dehydrogenases from residues G105 through G116, and A178 through
G197. PFAM identifies significant sequence identity with short
chain alcohol dehydrogenases. HSFM-2 contains the canonical
AMP-binding domain of short chain alcohol dehydrogenases at
residues 35-41. As shown in FIGS. 4A and 4B, HSFM-2 has chemical
and structural similarity with human 2,4-dienoyl-CoA reductase (GI
1575000; SEQ ID NO:19). In particular, HSFM-2 and human
2,4-dienoyl-CoA reductase share 31% identity. HSFM-2 and human
2,4-dienoyl-CoA reductase share canonical AMP-binding domains and
modified catalytic domains lacking the tyrosine residue (178-182
and 210-214 in HSFM-2 and human 2,4-dienoyl-CoA reductase,
respectively). A region of unique sequence in HSFM-2 from about
amino acid 149 to about amino acid 155 is encoded by a fragment of
SEQ ID NO:4 from about nucleotide 539 to about nucleotide. Northern
analysis shows the expression of this sequence in various
libraries, at least 75% of which are proliferative or cancerous, at
least 29% involve immune response. Of particular note is the
expression of HSFM-2 in gastrointestinal, reproductive, and nervous
tissues.
[0071] The invention also encompasses HSFM variants. A preferred
HSFM variant is one which has at least about 80%, more preferably
at least about 90%, and most preferably at least about 95% amino
acid sequence identity to the HSFM amino acid sequence, and which
contains at least one functional or structural characteristic of
HSFM.
[0072] The invention also encompasses polynucleotides which encode
HSFM. In a particular embodiment, the invention encompasses a
polynucleotide sequence comprising the sequence of SEQ ID NO:3,
which encodes an HSFM-1 as shown in FIGS. 1A, 1B, 1C, and 1D. In a
further embodiment, the invention encompasses the polynucleotide
sequence comprising the sequence of SEQ ID NO:4, which encodes an
HSFM-2 as shown in FIGS. 2A, 2B, 2C, 2D, and 2E.
[0073] The invention also encompasses a variant of a polynucleotide
sequence encoding HSFM. In particular, such a variant
polynucleotide sequence will have at least about 70%, more
preferably at least about 85%, and most preferably at least about
95% polynucleotide sequence identity to the polynucleotide sequence
encoding HSFM. A particular aspect of the invention encompasses a
variant of SEQ ID NO:3 which has at least about 70%, more
preferably at least about 85%, and most preferably at least about
95% polynucleotide sequence identity to SEQ ID NO:3. The invention
further encompasses a polynucleotide variant of SEQ ID NO:4 having
at least about 70%, more preferably at least about 85%, and most
preferably at least about 95% polynucleotide sequence identity to
SEQ ID NO:4. Any one of the polynucleotide variants described above
can encode an amino acid sequence which contains at least one
functional or structural characteristic of HSFM.
[0074] It will be appreciated by those skilled in the art that as a
result of the degeneracy of the genetic code, a multitude of
polynucleotide sequences encoding HSFM, some bearing minimal
similarity to the polynucleotide sequences of any known and
naturally occurring gene, may be produced. Thus, the invention
contemplates each and every possible variation of polynucleotide
sequence that could be made by selecting combinations based on
possible codon choices. These combinations are made in accordance
with the standard triplet genetic code as applied to the
polynucleotide sequence of naturally occurring HSFM, and all such
variations are to be considered as being specifically
disclosed.
[0075] Although nucleotide sequences which encode HSFM and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring HSFM under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding HSFM possessing a
substantially different codon usage, e.g., inclusion of
non-naturally occurring codons. Codons may be selected to increase
the rate at which expression of the peptide occurs in a particular
prokaryotic or eukaryotic host in accordance with the frequency
with which particular codons are utilized by the host. Other
reasons for substantially altering the nucleotide sequence encoding
HSFM and its derivatives without altering the encoded amino acid
sequences include the production of RNA transcripts having more
desirable properties, such as a greater half-life, than transcripts
produced from the naturally occurring sequence.
[0076] The invention also encompasses production of DNA sequences
which encode HSFM and HSFM derivatives, or fragments thereof,
entirely by synthetic chemistry. After production, the synthetic
sequence may be inserted into any of the many available expression
vectors and cell systems using reagents well known in the art.
Moreover, synthetic chemistry may be used to introduce mutations
into a sequence encoding HSFM or any fragment thereof.
[0077] Also encompassed by the invention are polynucleotide
sequences that are capable of hybridizing to the claimed
polynucleotide sequences, and, in particular, to those shown in SEQ
ID NO:3, SEQ ID NO:4, a fragment of SEQ ID NO:3, or a fragment of
SEQ ID NO:4, under various conditions of stringency. (See, e.g.,
Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399-407;
Kimmel, A. R. (1987) Methods Enzymol. 152:507-511.) For example,
stringent salt concentration will ordinarily be less than about 750
mM NaCl and 75 mM trisodium citrate, preferably less than about 500
mM NaCl and 50 mM trisodium citrate, and most preferably less than
about 250 mM NaCl and 25 mM trisodium citrate. Low stringency
hybridization can be obtained in the absence of organic solvent,
e.g., formamide, while high stringency hybridization can be
obtained in the presence of at least about 35% formamide, and most
preferably at least about 50% formamide. Stringent temperature
conditions will ordinarily include temperatures of at least about
30.degree. C., more preferably of at least about 37.degree. C., and
most preferably of at least about 42.degree. C. Varying additional
parameters, such as hybridization time, the concentration of
detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or
exclusion of carrier DNA, are well known to those skilled in the
art. Various levels of stringency are accomplished by combining
these various conditions as needed. In a preferred embodiment,
hybridization will occur at 30.degree. C. in 750 mM NaCl, 75 mM
trisodium citrate, and 1% SDS. In a more preferred embodiment,
hybridization will occur at 37.degree. C. in 500 mM NaCl, 50 mM
trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml
denatured salmon sperm DNA (ssDNA). In a most preferred embodiment,
hybridization will occur at 42.degree. C. in 250 mM NaCl, 25 mM
trisodium citrate, 1% SDS, 50% formamide, and 200 .mu.g/ml ssDNA.
Useful variations on these conditions will be readily apparent to
those skilled in the art.
[0078] The washing steps which follow hybridization can also vary
in stringency. Wash stringency conditions can be defined by salt
concentration and by temperature. As above, wash stringency can be
increased by decreasing salt concentration or by increasing
temperature. For example, stringent salt concentration for the wash
steps will preferably be less than about 30 mM NaCl and 3 mM
trisodium citrate, and most preferably less than about 15 mM NaCl
and 1.5 mM trisodium citrate. Stringent temperature conditions for
the wash steps will ordinarily include temperature of at least
about 25.degree. C., more preferably of at least about 42.degree.
C., and most preferably of at least about 68.degree. C. In a
preferred embodiment, wash steps will occur at 25.degree. C. in 30
mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred
embodiment, wash steps will occur at 42.degree. C. in 15 mM NaCl,
1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred
embodiment, wash steps will occur at 68.degree. C. in 15 mM NaCl,
1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on
these conditions will be readily apparent to those skilled in the
art.
[0079] Methods for DNA sequencing and analysis are well known in
the art. The methods may employ such enzymes as the Klenow fragment
of DNA polymerase I, SEQUENASE.RTM. (Amersham Pharmacia Biotech
Ltd., Uppsala, Sweden), Taq polymerase (The Perkin-Elmer Corp.,
Norwalk, Conn.), thermostable T7 polymerase (Amersham Pharmacia
Biotech Ltd., Uppsala, Sweden), or combinations of polymerases and
proofreading exonucleases, such as those found in the ELONGASE.TM.
amplification system (Life Technologies, Inc., Rockville, Md.).
Preferably, sequence preparation is automated with machines, e.g.,
the ABI CATALYST.TM. 800 (The Perkin-Elmer Corp., Norwalk, Conn.)
or MICROLAB.RTM. 2200 (Hamilton Co., Reno, Nev.) systems, in
combination with thermal cyclers. Sequencing can also be automated,
such as by ABI PRISM.TM. 373 or 377 systems (The Perkin Elmer
Corp., Norwalk, Conn.) or the MEGABACE.TM. 1000 capillary
electrophoresis system (Molecular Dynamics, Inc., Sunnyvale,
Calif.). Sequences can be analyzed using computer programs and
algorithms well known in the art. (See, e.g., Ausubel, supra, unit
7.7; and Meyers, R. A. (1995) Molecular Biology and Biotechnology,
Wiley VCH, Inc, New York, N.Y.)
[0080] The nucleic acid sequences encoding HSFM may be extended
utilizing a partial nucleotide sequence and employing various
PCR-based methods known in the art to detect upstream sequences,
such as promoters and regulatory elements. For example, one method
which may be employed, restriction-site PCR, uses universal and
nested primers to amplify unknown sequence from genomic DNA within
a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.) Another method, inverse PCR, uses primers that extend
in divergent directions to amplify unknown sequence from a
circularized template. The template is derived from restriction
fragments comprising a known genomic locus and surrounding
sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res.
16:8186.) A third method, capture PCR, involves PCR amplification
of DNA fragments adjacent to known sequences in human and yeast
artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991)
PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme digestions and ligations may be used to insert
an engineered double-stranded sequence into a region of unknown
sequence before performing PCR. Other methods which may be used to
retrieve unknown sequences are known in the art. (See, e.g.,
Parker. J. D. et al. (1991) Nucleic Acids Res. 19:3055-306).
Additionally, one may use PCR, nested primers, and
PromoterFinder.TM. libraries to walk genomic DNA (Clontech, Palo
Alto, Calif.). This procedure avoids the need to screen libraries
and is useful in finding intron/exon junctions. For all PCR-based
methods, primers may be designed using commercially available
software, such as OLIGO.TM. 4.06 Primer Analysis software (National
Biosciences Inc., Plymouth, Minn.) or another appropriate program,
to be about 22 to 30 nucleotides in length, to have a GC content of
about 50% or more, and to anneal to the template at temperatures of
about 68.degree. C. to 72.degree. C.
[0081] When screening for full-length cDNAs, it is preferable to
use libraries that have been size-selected to include larger cDNAs.
In addition, random-primed libraries, which often include sequences
containing the 5' regions of genes, are preferable for situations
in which an oligo d(T) library does not yield a full-length cDNA.
Genomic libraries may be useful for extension of sequence into 5'
non-transcribed regulatory regions.
[0082] Capillary electrophoresis systems which are commercially
available may be used to analyze the size or confirm the nucleotide
sequence of sequencing or PCR products. In particular, capillary
sequencing may employ flowable polymers for electrophoretic
separation, four different nucleotide-specific, laser-stimulated
fluorescent dyes, and a charge coupled device camera for detection
of the emitted wavelengths. Output/light intensity may be converted
to electrical signal using appropriate software (e.g.,
Genotyper.TM. and Sequence Navigator.TM., (The Perkin-Elmer Corp.,
Norwalk, Conn.)), and the entire process from loading of samples to
computer analysis and electronic data display may be computer
controlled. Capillary electrophoresis is especially preferable for
sequencing small DNA fragments which may be present in limited
amounts in a particular sample.
[0083] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode HSFM may be cloned in
recombinant DNA molecules that direct expression of HSFM, or
fragments or functional equivalents thereof, in appropriate host
cells. Due to the inherent degeneracy of the genetic code, other
DNA sequences which encode substantially the same or a functionally
equivalent amino acid sequence may be produced and used to express
HSFM.
[0084] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter HSFM-encoding sequences for a variety of purposes including,
but not limited to, modification of the cloning, processing, and/or
expression of the gene product. DNA shuffling by random
fragmentation and PCR reassembly of gene fragments and synthetic
oligonucleotides may be used to engineer the nucleotide sequences.
For example, oligonucleotide-mediated site-directed mutagenesis may
be used to introduce mutations that create new restriction sites,
alter glycosylation patterns, change codon preference, produce
splice variants, and so forth.
[0085] In another embodiment, sequences encoding HSFM may be
synthesized, in whole or in part, using chemical methods well known
in the art. (See, e.g., Caruthers, M. H. et al. (1980) Nucl. Acids
Res. Symp. Ser. 215-223, and Hom, T. et al. (1980) Nucl. Acids Res.
Symp. Ser. 225-232.) Alternatively, HSFM itself or a fragment
thereof may be synthesized using chemical methods. For example,
peptide synthesis can be performed using various solid-phase
techniques. (See, e.g., Roberge, J. Y. et al. (1995) Science
269:202-204.) Automated synthesis may be achieved using the ABI
431A Peptide Synthesizer (The Perkin-Elmer Corp., Norwalk, Conn.).
Additionally, the amino acid sequence of HSFM, or any part thereof,
may be altered during direct synthesis and/or combined with
sequences from other proteins, or any part thereof, to produce a
variant polypeptide.
[0086] The peptide may be substantially purified by preparative
high performance liquid chromatography. (See, e.g, Chiez, R. M. and
F. Z. Regnier (1990) Methods Enzymol. 182:392-421.) The composition
of the synthetic peptides may be confirmed by amino acid analysis
or by sequencing. (See, e.g., Creighton, T. (1984) Proteins,
Structures and Molecular Properties, W H Freeman and Co., New York,
N.Y.)
[0087] In order to express a biologically active HSFM, the
nucleotide sequences encoding HSFM or derivatives thereof may be
inserted into an appropriate expression vector, i.e., a vector
which contains the necessary elements for transcriptional and
translational control of the inserted coding sequence in a suitable
host. These elements include regulatory sequences, such as
enhancers, constitutive and inducible promoters, and 5' and 3'
untranslated regions in the vector and in polynucleotide sequences
encoding HSFM. Such elements may vary in their strength and
specificity. Specific initiation signals may also be used to
achieve more efficient translation of sequences encoding HSFM. Such
signals include the ATG initiation codon and adjacent sequences,
e.g. the Kozak sequence. In cases where sequences encoding HSFM and
its initiation codon and upstream regulatory sequences are inserted
into the appropriate expression vector, no additional
transcriptional or translational control signals may be needed.
However, in cases where only coding sequence, or a fragment
thereof, is inserted, exogenous translational control signals
including an in-frame ATG initiation codon should be provided by
the vector. Exogenous translational elements and initiation codons
may be of various origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
enhancers appropriate for the particular host cell system used.
(See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ.
20:125-162.)
[0088] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding HSFM and appropriate transcriptional and translational
control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview,
N.Y., ch. 4, 8, and 16-17; and Ausubel, F. M. et al. (1995, and
periodic supplements) Current Protocols in Molecular Biology, John
Wiley & Sons, New York, N.Y., ch. 9, 13, and 16.)
[0089] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding HSFM. These include, but
are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with viral expression vectors (e.g.,
baculovirus); plant cell systems transformed with viral expression
vectors (e.g., cauliflower mosaic virus (CAMV) or tobacco mosaic
virus (TMV)) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems. The invention is not
limited by the host cell employed.
[0090] In bacterial systems, a number of cloning and expression
vectors may be selected depending upon the use intended for
polynucleotide sequences encoding HSFM. For example, routine
cloning, subcloning, and propagation of polynucleotide sequences
encoding HSFM can be achieved using a multifunctional E. coli
vector such as Bluescript.RTM.) (Stratagene) or pSport1.TM. plasmid
(GIBCO BRL). Ligation of sequences encoding HSFM into the vector's
multiple cloning site disrupts the lacZ gene, allowing a
colorimetric screening procedure for identification of transformed
bacteria containing recombinant molecules. In addition, these
vectors may be useful for in vitro transcription, dideoxy
sequencing, single strand rescue with helper phage, and creation of
nested deletions in the cloned sequence. (See, e.g., Van Heeke, G.
and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When large
quantities of HSFM are needed, e.g. for the production of
antibodies, vectors which direct high level expression of HSFM may
be used. For example, vectors containing the strong, inducible T5
or T7 bacteriophage promoter may be used.
[0091] Yeast expression systems may be used for production of HSFM.
A number of vectors containing constitutive or inducible promoters,
such as alpha factor, alcohol oxidase, and PGH, may be used in the
yeast Saccharomyces cerevisiae or Pichia pastoris. In addition,
such vectors direct either the secretion or intracellular retention
of expressed proteins and enable integration of foreign sequences
into the host genome for stable propagation. (See, e.g., Ausubel,
supra; and Grant et al. (1987) Methods Enzymol. 153:516-54; Scorer,
C. A. et al. (1994) Bio/Technology 12:181-184.) Plant systems may
also be used for expression of HSFM. Transcription of sequences
encoding HSFM may be driven viral promoters, e.g., the 35S and 19S
promoters of CaMV used alone or in combination with the omega
leader sequence from TMV. (Takamatsu, N. (1987) EMBO J. 6:307-311.)
Alternatively, plant promoters such as the small subunit of RUBISCO
or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al.
(1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science
224:838-843; and Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105.) These constructs can be introduced into plant
cells by direct DNA transformation or pathogen-mediated
transfection. (See, e.g., Hobbs, S. or Murry, L. E. in McGraw Hill
Yearbook of Science and Technology (1992) McGraw Hill, New York,
N.Y.; pp. 191-196.)
[0092] In mammalian cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding HSFM may be ligated into an
adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader sequence. Insertion in a
non-essential E1 or E3 region of the viral genome may be used to
obtain infective virus which expresses HSFM in host cells. (See,
e.g., Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci.
81:3655-3659.) In addition, transcription enhancers, such as the
Rous sarcoma virus (RSV) enhancer, may be used to increase
expression in mammalian host cells. SV40 or EBV-based vectors may
also be used for high-level protein expression.
[0093] Human artificial chromosomes (HACs) may also be employed to
deliver larger fragments of DNA than can be contained in and
expressed from a plasmid. HACs of about 6 kb to 10 Mb are
constructed and delivered via conventional delivery methods
(liposomes, polycationic amino polymers, or vesicles) for
therapeutic purposes.
[0094] For long term production of recombinant proteins in
mammalian systems, stable expression of HSFM in cell lines is
preferred. For example, sequences encoding HSFM can be transformed
into cell lines using expression vectors which may contain viral
origins of replication and/or endogenous expression elements and a
selectable marker gene on the same or on a separate vector.
Following the introduction of the vector, cells may be allowed to
grow for about 1 to 2 days in enriched media before being switched
to selective media. The purpose of the selectable marker is to
confer resistance to a selective agent, and its presence allows
growth and recovery of cells which successfully express the
introduced sequences. Resistant clones of stably transformed cells
may be propagated using tissue culture techniques appropriate to
the cell type.
[0095] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase and adenine
phosphoribosyltransferase genes, for use in tk or apr cells,
respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232;
and Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite,
antibiotic, or herbicide resistance can be used as the basis for
selection. For example, dhfr confers resistance to methotrexate;
neo confers resistance to the aminoglycosides neomycin and G418;
and als or pat confer resistance to chlorsulfuron and
phosphinotricin acetyltransferase, respectively. (See, e.g.,
Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570;
Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14; and Murry,
supra.) Additional selectable genes have been described, e.g., trpB
and hisD, which alter cellular requirements for metabolites. (See,
e.g., Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad.
Sci. 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins (GFP) (Clontech, Palo Alto, Calif.), .beta.
glucuronidase and its substrate .beta.-D-glucuronoside, or
luciferase and its substrate luciferin may be used. These markers
can be used not only to identify transformants, but also to
quantify the amount of transient or stable protein expression
attributable to a specific vector system. (See, e.g., Rhodes, C. A.
et al. (1995) Methods Mol. Biol. 55:121-131.)
[0096] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, the presence
and expression of the gene may need to be confirmed. For example,
if the sequence encoding HSFM is inserted within a marker gene
sequence, transformed cells containing sequences encoding HSFM can
be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a
sequence encoding HSFM under the control of a single promoter.
Expression of the marker gene in response to induction or selection
usually indicates expression of the tandem gene as well.
[0097] In general, host cells that contain the nucleic acid
sequence encoding HSFM and that express HSFM may be identified by a
variety of procedures known to those of skill in the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA
hybridizations, PCR amplification, and protein bioassay or
immunoassay techniques which include membrane, solution, or chip
based technologies for the detection and/or quantification of
nucleic acid or protein sequences.
[0098] Immunological methods for detecting and measuring the
expression of HSFM using either specific polyclonal or monoclonal
antibodies are known in the art. Examples of such techniques
include enzyme-linked immunosorbent assays (ELISAs),
radioimmunoassays (RIAs), and fluorescence activated cell sorting
(FACS). A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on
HSFM is preferred, but a competitive binding assay may be employed.
These and other assays are well known in the art. (See, e.g.,
Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual,
APS Press, St Paul, Minn., Section IV; Coligan, J. E. et al. (1997
and periodic supplements) Current Protocols in Immunology, Greene
Pub. Associates and Wiley-Interscience, New York, N.Y.; and Maddox,
D. E. et al. (1983) J. Exp. Med. 158:1211-1216).
[0099] A wide variety of labels and conjugation techniques are
known by those skilled in the art and may be used in various
nucleic acid and amino acid assays. Means for producing labeled
hybridization or PCR probes for detecting sequences related to
polynucleotides encoding HSFM include oligolabeling, nick
translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding HSFM, or any
fragments thereof, may be cloned into a vector for the production
of an mRNA probe. Such vectors are known in the art, are
commercially available, and may be used to synthesize RNA probes in
vitro by addition of an appropriate RNA polymerase such as T7, T3,
or SP6 and labeled nucleotides. These procedures may be conducted
using a variety of commercially available kits, such as those
provided by Pharmacia & Upjohn (Kalamazoo, Mich.), Promega
(Madison, Wis.), and U.S. Biochemical Corp. (Cleveland, Ohio).
Suitable reporter molecules or labels which may be used for ease of
detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic agents, as well as substrates,
cofactors, inhibitors, magnetic particles, and the like.
[0100] Host cells transformed with nucleotide sequences encoding
HSFM may be cultured under conditions suitable for the expression
and recovery of the protein from cell culture. The protein produced
by a transformed cell may be secreted or retained intracellularly
depending on the sequence and/or the vector used. As will be
understood by those of skill in the art, expression vectors
containing polynucleotides which encode HSFM may be designed to
contain signal sequences which direct secretion of HSFM through a
prokaryotic or eukaryotic cell membrane.
[0101] In addition, a host cell strain may be chosen for its
ability to modulate expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be used to specify
protein targeting, folding, and/or activity. Different host cells
which have specific cellular machinery and characteristic
mechanisms for post-translational activities (e.g., CHO, HeLa,
MDCK, HEK293, and W138), are available from the American Type
Culture Collection (ATCC, Bethesda, Md.) and may be chosen to
ensure the correct modification and processing of the foreign
protein.
[0102] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding HSFM may be ligated
to a heterologous sequence resulting in translation of a fusion
protein in any of the aforementioned host systems. For example, a
chimeric HSFM protein containing a heterologous moiety that can be
recognized by a commercially available antibody may facilitate the
screening of peptide libraries for inhibitors of HSFM activity.
Heterologous protein and peptide moieties may also facilitate
purification of fusion proteins using commercially available
affinity matrices. Such moieties include, but are not limited to,
glutathione S-transferase (GST), maltose binding protein (MBP),
thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG,
c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification of their cognate fusion proteins on immobilized
glutathione, maltose, phenylarsine oxide, calmodulin, and
metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin
(HA) enable immunoaffinity purification of fusion proteins using
commercially available monoclonal and polyclonal antibodies that
specifically recognize these epitope tags. A fusion protein may
also be engineered to contain a proteolytic cleavage site located
between the HSFM encoding sequence and the heterologous protein
sequence, so that HSFM may be cleaved away from the heterologous
moiety following purification. Methods for fusion protein
expression and purification are discussed in Ausubel, F. M. et al.
(1995 and periodic supplements) Current Protocols in Molecular
Biology, John Wiley & Sons, New York, N.Y., ch 10. A variety of
commercially available kits may also be used to facilitate
expression and purification of fusion proteins.
[0103] In a further embodiment of the invention, synthesis of
radiolabeled HSFM may be achieved in vitro using the TNT.TM. rabbit
reticulocyte lysate or wheat germ extract systems (Promega,
Madison, Wis.). These systems couple transcription and translation
of protein-coding sequences operably associated with the T7, T3, or
SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid precursor, preferably
.sup.35S-methionine.
[0104] Fragments of HSFM may be produced not only by recombinant
production, but also by direct peptide synthesis using solid-phase
techniques. (See, e.g., Creighton, supra pp. 55-60.) Protein
synthesis may be performed by manual techniques or by automation.
Automated synthesis may be achieved, for example, using the Applied
Biosystems 431 A Peptide Synthesizer (The Perkin-Elmer Corp.,
Norwalk, Conn.). Various fragments of HSFM may be synthesized
separately and then combined to produce the full length
molecule.
[0105] Therapeutics
[0106] Chemical and structural similarity exists among HSFM-1 and
human 1-cis-retinol dehydrogenase (GI 1616654) and rat retinol
dehydrogenase (GI 841197). In addition, HSFM-1 is expressed in
proliferative and inflamed tissues. Therefore, HSFM-1 appears to
play a role in disorders associated with cell proliferation,
inflammation, and fatty acid and steroid metabolism.
[0107] Chemical and structural similarity exists between HSFM-2 and
human 2,4-dienoyl-CoA reductase (GI 1575000). In addition, HSFM-2
is expressed in proliferative and inflamed tissues. Therefore,
HSFM-2 appears to play a role in disorders associated with
proliferation, inflammation, and fatty acid and steroid
metabolism.
[0108] Therefore, in one embodiment, HSFM or a fragment or
derivative thereof may be administered to a subject to treat or
prevent a cell proliferative disorder. Such cell proliferative
disorders can include, but are not limited to, actinic keratosis,
arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis,
mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal
nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary
thrombocythemia, and cancers including adenocarcinoma, leukemia,
lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in
particular, cancers of the adrenal gland, bladder, bone, bone
marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus.
[0109] In another embodiment, a vector capable of expressing HSFM
or a fragment or derivative thereof may be administered to a
subject to treat or prevent a cell proliferative disorder
including, but not limited to, those described above.
[0110] In a further embodiment, a pharmaceutical composition
comprising a substantially purified HSFM in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a cell proliferative disorder including, but not
limited to, those provided above.
[0111] In still another embodiment, an agonist which modulates the
activity of HSFM may be administered to a subject to treat or
prevent a cell proliferative disorder including, but not limited
to, those listed above.
[0112] In another embodiment, HSFM or a fragment or derivative
thereof may be administered to a subject to treat or prevent an
inflammatory disorder. Such inflammatory disorders can include, but
are not limited to, acquired immunodeficiency syndrome (AIDS),
Addison's disease, adult respiratory distress syndrome, allergies,
ankylosing spondylitis, amyloidosis, anemia, asthma, autoimmune
hemolytic anemia, autoimmune thyroiditis, bronchitis,
cholecystitis, contact dermatitis, Crohn's disease, atopic
dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic
lymphopenia with lymphocytotoxins, erythroblastosis fetalis,
erythema nodosum, atrophic gastritis, glomerulonephritis,
Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple
sclerosis, myasthenia gravis, myocardial or pericardial
inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, Reiter's syndrome, rheumatoid arthritis, scleroderma,
Sjogren's syndrome, systemic anaphylaxis, systemic lupus
erythematosus, systemic sclerosis, thrombocytopenic purpura,
ulcerative colitis, uveitis, Werner syndrome, complications of
cancer, hemodialysis, and extracorporeal circulation, viral,
bacterial, fungal, parasitic, protozoal, and helminthic infections,
and trauma.
[0113] In another embodiment, a vector capable of expressing HSFM
or a fragment or derivative thereof may be administered to a
subject to treat or prevent an inflammatory disorder including, but
not limited to, those described above.
[0114] In a further embodiment, a pharmaceutical composition
comprising a substantially purified HSFM in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent an inflammatory disorder including, but not
limited to, those provided above.
[0115] In still another embodiment, an agonist which modulates the
activity of HSFM may be administered to a subject to treat or
prevent an inflammatory disorder including, but not limited to,
those listed above.
[0116] In another embodiment, HSFM or a fragment or derivative
thereof may be administered to a subject to treat or prevent a
fatty acid and steroid metabolic disorder. Such fatty acid and
steroid metabolic disorders can include, but are not limited to,
fatty hepatocirrhosis, hyperadrenalism, hypoadrenalism,
hyperparathyroidism, hypoparathyroidism, hypercholesterolemia,
hyperthyroidism, hypothyroidism, hyperlipidemia, hyperlipemia,
lipid myopathies, obesity, lipodystrophies, 2,4-dienoyl-CoA
reductase deficiency, acyl-CoA oxidase deficiency, thiolase
deficiency, peroxisomal bifunctional protein deficiency,
mitochondrial carnitine palmitoyl transferase and carnitine
deficiency, mitochondrial very-long-chain acyl-CoA dehydrogenase
deficiency, mitochondrial medium-chain acyl-CoA dehydrogenase
deficiency, mitochondrial short-chain acyl-CoA dehydrogenase
deficiency, mitochondrial electron transport flavoprotein and
electron transport flavoprotein:ubiquinone oxidoreductase
deficiency, mitochondrial trifunctional protein deficiency, and
mitochondrial short-chain 3-hydroxyacyl-CoA dehydrogenase
deficiency.
[0117] In another embodiment, a vector capable of expressing HSFM
or a fragment or derivative thereof may be administered to a
subject to treat or prevent a fatty acid and steroid metabolic
disorder including, but not limited to, those described above.
[0118] In a further embodiment, a pharmaceutical composition
comprising a substantially purified HSFM in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a fatty acid and steroid metabolic disorder
including, but not limited to, those provided above.
[0119] In still another embodiment, an agonist which modulates the
activity of HSFM may be administered to a subject to treat or
prevent a fatty acid and steroid metabolic disorder including, but
not limited to, those listed above.
[0120] In other embodiments, any of the proteins, antagonists,
antibodies, agonists, complementary sequences, or vectors of the
invention may be administered in combination with other appropriate
therapeutic agents. Selection of the appropriate agents for use in
combination therapy may be made by one of ordinary skill in the
art, according to conventional pharmaceutical principles. The
combination of therapeutic agents may act synergistically to effect
the treatment or prevention of the various disorders described
above. Using this approach, one may be able to achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the
potential for adverse side effects.
[0121] An antagonist of HSFM may be produced using methods which
are generally known in the art. In particular, purified HSFM may be
used to produce antibodies or to screen libraries of pharmaceutical
agents to identify those which specifically bind HSFM. Antibodies
to HSFM may also be generated using methods that are well known in
the art. Such antibodies may include, but are not limited to,
polyclonal, monoclonal, chimeric, and single chain antibodies. Fab
fragments, and fragments produced by a Fab expression library.
Neutralizing antibodies (i.e., those which inhibit dimer formation)
are especially preferred for therapeutic use.
[0122] For the production of polyclonal antibodies, various hosts
including goats, rabbits, rats, mice, humans, and others may be
immunized by injection with HSFM or with any fragment or
oligopeptide thereof which has immunogenic properties. Rats and
mice are preferred hosts for downstream applications involving
monoclonal antibody production. Depending on the host species,
various adjuvants may be used to increase immunological response.
Such adjuvants include, but are not limited to, Freund's, mineral
gels such as aluminum hydroxide, and surface active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, KLH, and dinitrophenol. Among adjuvants used in humans,
BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are
especially preferable. (For review of methods for antibody
production and analysis, see, e.g., Harlow, E. and Lane, D. (1988)
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y.)
[0123] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to HSFM have an amino acid
sequence consisting of at least about 5 amino acids, and, more
preferably, of at least about 14 amino acids. It is also preferable
that these oligopeptides, peptides, or fragments are identical to a
portion of the amino acid sequence of the natural protein and
contain the entire amino acid sequence of a small, naturally
occurring molecule. Short stretches of HSFM amino acids may be
fused with those of another protein, such as KLH, and antibodies to
the chimeric molecule may be produced.
[0124] Monoclonal antibodies to HSFM may be prepared using any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma
technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G.
et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl.
Acad. Sci. 80:2026-2030; and Cole, S. P. et al. (1984) Mol. Cell
Biol. 62:109-120.)
[0125] In addition, techniques developed for the production of
"chimeric antibodies," such as the splicing of mouse antibody genes
to human antibody genes to obtain a molecule with appropriate
antigen specificity and biological activity, can be used. (See,
e.g., Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci.
81:6851-6855; Neuberger, M. S. et al. (1984) Nature 312:604-608;
and Takeda, S. et al. (1985) Nature 314:452-454.) Alternatively,
techniques described for the production of single chain antibodies
may be adapted, using methods known in the art, to produce
HSFM-specific single chain antibodies. Antibodies with related
specificity, but of distinct idiotypic composition, may be
generated by chain shuffling from random combinatorial
immunoglobulin libraries. (See, e.g., Burton D. R. (1991) Proc.
Natl. Acad. Sci. 88:10134-10137.)
[0126] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in the literature. (See, e.g., Orlandi, R. et
al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; and Winter, G. et
al. (1991) Nature 349:293-299.)
[0127] Antibody fragments which contain specific binding sites for
HSFM may also be generated. For example, such fragments include,
but are not limited to, F(ab).sub.2 fragments produced by pepsin
digestion of the antibody molecule and Fab fragments generated by
reducing the disulfide bridges of the F(ab).sub.2 fragments.
Alternatively, Fab expression libraries may be constructed to allow
rapid and easy identification of monoclonal Fab fragments with the
desired specificity. (See, e.g., Huse. W. D. et al. (1989) Science
246:1275-1281.)
[0128] Various immunoassays may be used for screening to identify
antibodies having the desired specificity and minimal
cross-reactivity. Numerous protocols for competitive binding or
immunoradiometric assays using either polyclonal or monoclonal
antibodies with established specificities are well known in the
art. Such immunoassays typically involve the measurement of complex
formation between HSFM and its specific antibody. A two-site,
monoclonal-based immunoassay utilizing monoclonal antibodies
reactive to two non-interfering HSFM epitopes is preferred, but a
competitive binding assay may also be employed. (Maddox,
supra.)
[0129] Various methods such as Scatchard analysis in conjunction
with radioimmunoassay techniques may be used to assess the affinity
of antibodies for HSFM. Affinity is expressed as an association
constant, K.sub.a, which is defined as the molar concentration of
HSFM-antibody complex divided by the molar concentrations of free
antigen and free antibody under equilibrium conditions. The K.sub.a
determined for a preparation of polyclonal antibodies, which are
heterogeneous in their affinities for multiple HSFM epitopes,
represents the average affinity, or avidity, of the antibodies for
HSFM. The K.sub.a determined for a preparation of monoclonal
antibodies, which are monospecific for a particular HSFM epitope,
represents a true measure of affinity. High-affinity antibody
preparations with K.sub.a ranging from about 10.sup.9 to 10.sup.12
L/mole are preferred for use in immunoassays in which the
HSFM-antibody complex must withstand rigorous manipulations.
Low-affinity antibody preparations with K.sub.a ranging from about
10.sup.6 to 10.sup.7 L/mole are preferred for use in
immunopurification and similar procedures which ultimately require
dissociation of HSFM, preferably in active form, from the antibody.
(Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington, D.C.; and Liddell, J. E. and Cryer, A. (1991) A
Practical Guide to Monoclonal Antibodies, John Wiley & Sons,
New York, N.Y.)
[0130] The titre and avidity of polyclonal antibody preparations
may be further evaluated to determine the quality and suitability
of such preparations for certain downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2
mg specific antibody/ml, preferably 5-10 mg specific antibody/ml,
is preferred for use in procedures requiring precipitation of
HSFM-antibody complexes. Procedures for evaluating antibody
specificity, titer, and avidity, and guidelines for antibody
quality and usage in various applications, are generally available.
(See, e.g., Catty, supra, and Coligan et al. supra.)
[0131] In another embodiment of the invention, the polynucleotides
encoding HSFM, or any fragment or complement thereof, may be used
for therapeutic purposes. In one aspect, the complement of the
polynucleotide encoding HSFM may be used in situations in which it
would be desirable to block the transcription of the mRNA. In
particular, cells may be transformed with sequences complementary
to polynucleotides encoding HSFM. Thus, complementary molecules or
fragments may be used to modulate HSFM activity, or to achieve
regulation of gene function. Such technology is now well known in
the art, and sense or antisense oligonucleotides or larger
fragments can be designed from various locations along the coding
or control regions of sequences encoding HSFM.
[0132] Expression vectors derived from retroviruses, adenoviruses,
or herpes or vaccinia viruses, or from various bacterial plasmids,
may be used for delivery of nucleotide sequences to the targeted
organ, tissue, or cell population. Methods which are well known to
those skilled in the art can be used to construct vectors to
express nucleic acid sequences complementary to the polynucleotides
encoding HSFM. (See, e.g., Sambrook, supra; and Ausubel,
supra.)
[0133] Genes encoding HSFM can be turned off by transforming a cell
or tissue with expression vectors which express high levels of a
polynucleotide, or fragment thereof, encoding HSFM. Such constructs
may be used to introduce untranslatable sense or antisense
sequences into a cell. Even in the absence of integration into the
DNA, such vectors may continue to transcribe RNA molecules until
they are disabled by endogenous nucleases. Transient expression may
last for a month or more with a non-replicating vector, and may
last even longer if appropriate replication elements are part of
the vector system.
[0134] As mentioned above, modifications of gene expression can be
obtained by designing complementary sequences or antisense
molecules (DNA, RNA, or PNA) to the control, 5, or regulatory
regions of the gene encoding HSFM. Oligonucleotides derived from
the transcription initiation site, e.g., between about positions
-10 and +10 from the start site, are preferred. Similarly,
inhibition can be achieved using triple helix base-pairing
methodology. Triple helix pairing is useful because it causes
inhibition of the ability of the double helix to open sufficiently
for the binding of polymerases, transcription factors, or
regulatory molecules. Recent therapeutic advances using triplex DNA
have been described in the literature. (See, e.g., Gee, J. E. et
al. (1994) in Huber, B. E. and B. I. Carr, Molecular and
Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y., pp.
163-177.) A complementary sequence or antisense molecule may also
be designed to block translation of mRNA by preventing the
transcript from binding to ribosomes.
[0135] Ribozymes, enzymatic RNA molecules, may also be used to
catalyze the specific cleavage of RNA. The mechanism of ribozyme
action involves sequence-specific hybridization of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic
cleavage. For example, engineered hammerhead motif ribozyme
molecules may specifically and efficiently catalyze endonucleolytic
cleavage of sequences encoding HSFM.
[0136] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites, including the following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15
and 20 ribonucleotides, corresponding to the region of the target
gene containing the cleavage site, may be evaluated for secondary
structural features which may render the oligonucleotide
inoperable. The suitability of candidate targets may also be
evaluated by testing accessibility to hybridization with
complementary oligonucleotides using ribonuclease protection
assays.
[0137] Complementary ribonucleic acid molecules and ribozymes of
the invention may be prepared by any method known in the art for
the synthesis of nucleic acid molecules. These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis.
[0138] Alternatively, RNA molecules may be generated by in vitro
and in vivo transcription of DNA sequences encoding HSFM. Such DNA
sequences may be incorporated into a wide variety of vectors with
suitable RNA polymerase promoters such as T7 or SP6.
[0139] Alternatively, these cDNA constructs that synthesize
complementary RNA, constitutively or inducibly, can be introduced
into cell lines, cells, or tissues.
[0140] RNA molecules may be modified to increase intracellular
stability and half-life. Possible modifications include, but are
not limited to, the addition of flanking sequences at the 5' and/or
3' ends of the molecule, or the use of phosphorothioate or 2'
O-methyl rather than phosphodiesterase linkages within the backbone
of the molecule. This concept is inherent in the production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine, queosine, and wybutosine, as
well as acetyl-, methyl-, thio-, and similarly modified forms of
adenine, cytidine, guanine, thymine, and uridine which are not as
easily recognized by endogenous endonucleases.
[0141] Many methods for introducing vectors into cells or tissues
are available and equally suitable for use in vivo, in vitro, and
ex vivo. For ex vivo therapy, vectors may be introduced into stem
cells taken from the patient and clonally propagated for autologous
transplant back into that same patient. Delivery by transfection,
by liposome injections, or by polycationic amino polymers may be
achieved using methods which are well known in the art. (See, e.g.,
Goldman, C. K. et al. (1997) Nature Biotechnology 15:462-466.)
[0142] Any of the therapeutic methods described above may be
applied to any subject in need of such therapy, including, for
example, mammals such as dogs, cats, cows, horses, rabbits,
monkeys, and most preferably, humans.
[0143] An additional embodiment of the invention relates to the
administration of a pharmaceutical or sterile composition, in
conjunction with a pharmaceutically acceptable carrier, for any of
the therapeutic effects discussed above. Such pharmaceutical
compositions may consist of HSFM, antibodies to HSFM, and mimetics,
agonists, antagonists, or inhibitors of HSFM. The compositions may
be administered alone or in combination with at least one other
agent, such as a stabilizing compound, which may be administered in
any sterile, biocompatible pharmaceutical carrier including, but
not limited to, saline, buffered saline, dextrose, and water. The
compositions may be administered to a patient alone, or in
combination with other agents, drugs, or hormones.
[0144] The pharmaceutical compositions utilized in this invention
may be administered by any number of routes including, but not
limited to, oral, intravenous, intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal,
subcutaneous, intraperitoneal, intranasal, enteral, topical,
sublingual, or rectal means.
[0145] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically-acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. Further details on techniques for
formulation and administration may be found in the latest edition
of Remington's Pharmaceutical Sciences (Maack Publishing Co.,
Easton, Pa.).
[0146] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral administration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for ingestion by the patient.
[0147] Pharmaceutical preparations for oral use can be obtained
through combining active compounds with solid excipient and
processing the resultant mixture of granules (optionally, after
grinding) to obtain tablets or dragee cores. Suitable auxiliaries
can be added, if desired. Suitable excipients include carbohydrate
or protein fillers, such as sugars, including lactose, sucrose,
mannitol, and sorbitol; starch from corn, wheat, rice, potato, or
other plants; cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
gums, including arabic and tragacanth; and proteins, such as
gelatin and collagen. If desired, disintegrating or solubilizing
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, and alginic acid or a salt thereof, such as
sodium alginate.
[0148] Dragee cores may be used in conjunction with suitable
coatings, such as concentrated sugar solutions, which may also
contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for product
identification or to characterize the quantity of active compound,
i.e., dosage.
[0149] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers
or binders, such as lactose or starches, lubricants, such as talc
or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid, or liquid
polyethylene glycol with or without stabilizers.
[0150] Pharmaceutical formulations suitable for parenteral
administration may be formulated in aqueous solutions, preferably
in physiologically compatible buffers such as Hanks's solution,
Ringer's solution, or physiologically buffered saline. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils, such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic
amino polymers may also be used for delivery. Optionally, the
suspension may also contain suitable stabilizers or agents to
increase the solubility of the compounds and allow for the
preparation of highly concentrated solutions.
[0151] For topical or nasal administration, penetrants appropriate
to the particular barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art.
[0152] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is known in the art, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping,
or lyophilizing processes.
[0153] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to,
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and
succinic acid. Salts tend to be more soluble in aqueous or other
protonic solvents than are the corresponding free base forms. In
other cases, the preferred preparation may be a lyophilized powder
which may contain any or all of the following: 1 mM to 50 mM
histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol, at a pH range
of 4.5 to 5.5, that is combined with buffer prior to use.
[0154] After pharmaceutical compositions have been prepared, they
can be placed in an appropriate container and labeled for treatment
of an indicated condition. For administration of HSFM, such
labeling would include amount, frequency, and method of
administration.
[0155] Pharmaceutical compositions suitable for use in the
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose.
The determination of an effective dose is well within the
capability of those skilled in the art.
[0156] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays, e.g., of
neoplastic cells or in animal models such as mice, rats, rabbits,
dogs, or pigs. An animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes
for administration in humans.
[0157] A therapeutically effective dose refers to that amount of
active ingredient, for example HSFM or fragments thereof,
antibodies of HSFM, and agonists, antagonists or inhibitors of
HSFM, which ameliorates the symptoms or condition. Therapeutic
efficacy and toxicity may be determined by standard pharmaceutical
procedures in cell cultures or with experimental animals, such as
by calculating the ED50 (the dose therapeutically effective in 50%
of the population) or LD.sub.50 (the dose lethal to 50% of the
population) statistics. The dose ratio of therapeutic to toxic
effects is the therapeutic index, and it can be expressed as the
ED.sub.50/LD.sub.50 ratio. Pharmaceutical compositions which
exhibit large therapeutic indices are preferred. The data obtained
from cell culture assays and animal studies are used to formulate a
range of dosage for human use. The dosage contained in such
compositions is preferably within a range of circulating
concentrations that includes the ED50 with little or no toxicity.
The dosage varies within this range depending upon the dosage form
employed, the sensitivity of the patient, and the route of
administration.
[0158] The exact dosage will be determined by the practitioner, in
light of factors related to the subject requiring treatment. Dosage
and administration are adjusted to provide sufficient levels of the
active moiety or to maintain the desired effect. Factors which may
be taken into account include the severity of the disease state,
the general health of the subject, the age, weight, and gender of
the subject, time and frequency of administration, drug
combination(s), reaction sensitivities, and response to therapy.
Long-acting pharmaceutical compositions may be administered every 3
to 4 days, every week, or biweekly depending on the half-life and
clearance rate of the particular formulation.
[0159] Normal dosage amounts may vary from about 0.1 .mu.g to
100,000 .mu.g, up to a total dose of about 1 gram, depending upon
the route of administration. Guidance as to particular dosages and
methods of delivery is provided in the literature and generally
available to practitioners in the art. Those skilled in the art
will employ different formulations for nucleotides than for
proteins or their inhibitors. Similarly, delivery of
polynucleotides or polypeptides will be specific to particular
cells, conditions, locations, etc.
[0160] Diagnostics
[0161] In another embodiment, antibodies which specifically bind
HSFM may be used for the diagnosis of disorders characterized by
expression of HSFM, or in assays to monitor patients being treated
with HSFM or agonists, antagonists, or inhibitors of HSFM.
Antibodies useful for diagnostic purposes may be prepared in the
same manner as described above for therapeutics. Diagnostic assays
for HSFM include methods which utilize the antibody and a label to
detect HSFM in human body fluids or in extracts of cells or
tissues. The antibodies may be used with or without modification,
and may be labeled by covalent or non-covalent attachment of a
reporter molecule. A wide variety of reporter molecules, several of
which are described above, are known in the art and may be
used.
[0162] A variety of protocols for measuring HSFM, including ELISAs,
RIAs, and FACS, are known in the art and provide a basis for
diagnosing altered or abnormal levels of HSFM expression. Normal or
standard values for HSFM expression are established by combining
body fluids or cell extracts taken from normal mammalian subjects,
preferably human, with antibody to HSFM under conditions suitable
for complex formation The amount of standard complex formation may
be quantitated by various methods, preferably by photometric means.
Quantities of HSFM expressed in subject, control, and disease
samples from biopsied tissues are compared with the standard
values. Deviation between standard and subject values establishes
the parameters for diagnosing disease.
[0163] In another embodiment of the invention, the polynucleotides
encoding HSFM may be used for diagnostic purposes. The
polynucleotides which may be used include oligonucleotide
sequences, complementary RNA and DNA molecules, and PNAs. The
polynucleotides may be used to detect and quantitate gene
expression in biopsied tissues in which expression of HSFM may be
correlated with disease. The diagnostic assay may be used to
determine absence, presence, and excess expression of HSFM, and to
monitor regulation of HSFM levels during therapeutic
intervention.
[0164] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding HSFM or closely related molecules may be used
to identify nucleic acid sequences which encode HSFM. The
specificity of the probe, whether it is made from a highly specific
region, e.g., the 5' regulatory region, or from a less specific
region, e.g., a conserved motif, and the stringency of the
hybridization or amplification (maximal, high, intermediate, or
low), will determine whether the probe identifies only naturally
occurring sequences encoding HSFM, allelic variants, or related
sequences.
[0165] Probes may also be used for the detection of related
sequences, and should preferably have at least 50% sequence
identity to any of the HSFM encoding sequences. The hybridization
probes of the subject invention may be DNA or RNA and may be
derived from the sequence of SEQ ID NO:3, SEQ ID NO:4, or from
genomic sequences including promoters, enhancers, and introns of
the HSFM gene.
[0166] Means for producing specific hybridization probes for DNAs
encoding HSFM include the cloning of polynucleotide sequences
encoding HSFM or HSFM derivatives into vectors for the production
of mRNA probes. Such vectors are known in the art, are commercially
available, and may be used to synthesize RNA probes in vitro by
means of the addition of the appropriate RNA polymerases and the
appropriate labeled nucleotides. Hybridization probes may be
labeled by a variety of reporter groups, for example, by
radionuclides such as .sup.32P or .sup.35S, or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin
coupling systems, and the like.
[0167] Polynucleotide sequences encoding HSFM may be used for the
diagnosis of a disorder associated with expression of HSFM.
Examples of such a disorder include, but are not limited to, cell
proliferative disorders such as actinic keratosis,
arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis,
mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal
nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary
thrombocythemia, and cancers including adenocarcinoma, leukemia,
lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in
particular, cancers of the adrenal gland, bladder, bone, bone
marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus; inflammatory disorders
such as acquired immunodeficiency syndrome (A/DS), Addison's
disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis, anemia, asthma, autoimmune hemolytic
anemia, autoimmune thyroiditis, bronchitis, cholecystitis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,
diabetes mellitus, emphysema, episodic lymphopenia with
lymphocytotoxins, erythroblastosis fetalis, erythema nodosum,
atrophic gastritis, glomerulonephritis, Goodpasture's syndrome,
gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,
irritable bowel syndrome, multiple sclerosis, myasthenia gravis,
myocardial or pericardial inflammation, osteoarthritis,
osteoporosis, pancreatitis, polymyositis, Reiter's syndrome,
rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial, fungal, parasitic, protozoal, and
helminthic infections, and trauma; and fatty acid and steroid
metabolic disorders such as fatty hepatocirrhosis, hyperadrenalism,
hypoadrenalism, hyperparathyroidism, hypoparathyroidism,
hypercholesterolemia, hyperthyroidism, hypothyroidism,
hyperlipidemia, hyperlipemia, lipid myopathies, obesity,
lipodystrophies, 2,4-dienoyl-CoA reductase deficiency, acyl-CoA
oxidase deficiency, thiolase deficiency, peroxisomal bifunctional
protein deficiency, mitochondrial carnitine palmitoyl transferase
and carnitine deficiency, mitochondrial very-long-chain acyl-CoA
dehydrogenase deficiency, mitochondrial medium-chain acyl-CoA
dehydrogenase deficiency, mitochondrial short-chain acyl-CoA
dehydrogenase deficiency, mitochondrial electron transport
flavoprotein and electron transport flavoprotein:ubiquinone
oxidoreductase deficiency, mitochondrial trifunctional protein
deficiency, and mitochondrial short-chain 3-hydroxyacyl-CoA
dehydrogenase deficiency. The polynucleotide sequences encoding
HSFM may be used in Southern or Northern analysis, dot blot, or
other membrane-based technologies; in PCR technologies; in
dipstick, pin, and ELISA assays; and in microarrays utilizing
fluids or tissues from patients to detect altered HSFM expression.
Such qualitative or quantitative methods are well known in the
art.
[0168] In a particular aspect, the nucleotide sequences encoding
HSFM may be useful in assays that detect the presence of associated
disorders, particularly those mentioned above. The nucleotide
sequences encoding HSFM may be labeled by standard methods and
added to a fluid or tissue sample from a patient under conditions
suitable for the formation of hybridization complexes. After a
suitable incubation period, the sample is washed and the signal is
quantitated and compared with a standard value. If the amount of
signal in the patient sample is significantly altered in comparison
to a control sample then the presence of altered levels of
nucleotide sequences encoding HSFM in the sample indicates the
presence of the associated disorder. Such assays may also be used
to evaluate the efficacy of a particular therapeutic treatment
regimen in animal studies, in clinical trials, or to monitor the
treatment of an individual patient.
[0169] In order to provide a basis for the diagnosis of a disorder
associated with expression of HSFM, a normal or standard profile
for expression is established. This may be accomplished by
combining body fluids or cell extracts taken from normal subjects,
either animal or human, with a sequence, or a fragment thereof,
encoding HSFM, under conditions suitable for hybridization or
amplification. Standard hybridization may be quantified by
comparing the values obtained from normal subjects with values from
an experiment in which a known amount of a substantially purified
polynucleotide is used. Standard values obtained in this manner may
be compared with values obtained from samples from patients who are
symptomatic for a disorder. Deviation from standard values is used
to establish the presence of a disorder.
[0170] Once the presence of a disorder is established and a
treatment protocol is initiated, hybridization assays may be
repeated on a regular basis to determine if the level of expression
in the patient begins to approximate that which is observed in the
normal subject. The results obtained from successive assays may be
used to show the efficacy of treatment over a period ranging from
several days to months.
[0171] With respect to cancer, the presence of a relatively high
amount of transcript in biopsied tissue from an individual may
indicate a predisposition for the development of the disease, or
may provide a means for detecting the disease prior to the
appearance of actual clinical symptoms. A more definitive diagnosis
of this type may allow health professionals to employ preventative
measures or aggressive treatment earlier thereby preventing the
development or further progression of the cancer.
[0172] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding HSFM may involve the use of PCR. These
oligomers may be chemically synthesized, generated enzymatically,
or produced in vitro. Oligomers will preferably contain a fragment
of a polynucleotide encoding HSFM, or a fragment of a
polynucleotide complementary to the polynucleotide encoding HSFM,
and will be employed under optimized conditions for identification
of a specific gene or condition. Oligomers may also be employed
under less stringent conditions for detection or quantitation of
closely related DNA or RNA sequences.
[0173] Methods which may also be used to quantitate the expression
of HSFM include radiolabeling or biotinylating nucleotides,
coamplification of a control nucleic acid, and interpolating
results from standard curves. (See, e.g., Melby, P. C. et al.
(1993) J. Immunol. Methods 159:235-244; and Duplaa, C. et al.
(1993) Anal. Biochem. 229-236.) The speed of quantitation of
multiple samples may be accelerated by running the assay in an
ELISA format where the oligomer of interest is presented in various
dilutions and a spectrophotometric or colorimetric response gives
rapid quantitation.
[0174] In further embodiments, oligonucleotides or longer fragments
derived from any of the polynucleotide sequences described herein
may be used as targets in a microarray. The microarray can be used
to monitor the expression level of large numbers of genes
simultaneously and to identify genetic variants, mutations, and
polymorphisms. This information may be used to determine gene
function, to understand the genetic basis of a disorder, to
diagnose a disorder, and to develop and monitor the activities of
therapeutic agents.
[0175] Microarrays may be prepared, used, and analyzed using
methods known in the art. (See, e.g., Brennan, T. M. et al. (1995)
U.S. Pat. No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad.
Sci. 93:10614-10619; Baldeschweiler et al. (1995) PCT application
WO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;
Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155;
and Heller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)
[0176] In another embodiment of the invention, nucleic acid
sequences encoding HSFM may be used to generate hybridization
probes useful in mapping the naturally occurring genomic sequence.
The sequences may be mapped to a particular chromosome, to a
specific region of a chromosome, or to artificial chromosome
constructions, e.g., human artificial chromosomes (HACs), yeast
artificial chromosomes (YACs), bacterial artificial chromosomes
(BACs), bacterial P1 constructions, or single chromosome cDNA
libraries. (See, e.g., Price, C. M. (1993) Blood Rev. 7:127-134;
and Trask, B. J. (1991) Trends Genet. 7:149-154.)
[0177] Fluorescent in situ hybridization (FISH) may be correlated
with other physical chromosome mapping techniques and genetic map
data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, R. A.
(ed.) Molecular Biology and Biotechnology, VCH Publishers New York,
N.Y., pp. 965-968.) Examples of genetic map data can be found in
various scientific journals or at the Online Mendelian Inheritance
in Man (OMIM) site. Correlation between the location of the gene
encoding HSFM on a physical chromosomal map and a specific
disorder, or a predisposition to a specific disorder, may help
define the region of DNA associated with that disorder. The
nucleotide sequences of the invention may be used to detect
differences in gene sequences among normal, carrier, and affected
individuals.
[0178] In situ hybridization of chromosomal preparations and
physical mapping techniques, such as linkage analysis using
established chromosomal markers, may be used for extending genetic
maps. Often the placement of a gene on the chromosome of another
mammalian species, such as mouse, may reveal associated markers
even if the number or arm of a particular human chromosome is not
known. New sequences can be assigned to chromosomal arms by
physical mapping. This provides valuable information to
investigators searching for disease genes using positional cloning
or other gene discovery techniques. Once the disease or syndrome
has been crudely localized by genetic linkage to a particular
genomic region, e.g., ataxia-telangiectasia to 11q22-23, any
sequences mapping to that area may represent associated or
regulatory genes for further investigation. (See, e.g., Gatti, R.
A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of
the subject invention may also be used to detect differences in the
chromosomal location due to translocation, inversion, etc., among
normal, carrier, or affected individuals.
[0179] In another embodiment of the invention, HSFM, its catalytic
or immunogenic fragments, or oligopeptides thereof can be used for
screening libraries of compounds in any of a variety of drug
screening techniques. The fragment employed in such screening may
be free in solution, affixed to a solid support, borne on a cell
surface, or located intracellularly. The formation of binding
complexes between HSFM and the agent being tested may be
measured.
[0180] Another technique for drug screening provides for high
throughput screening of compounds having suitable binding affinity
to the protein of interest. (See, e.g., Geysen, et al. (1984) PCT
application WO84/03564.) In this method, large numbers of different
small test compounds are synthesized on a solid substrate, such as
plastic pins or some other surface. The test compounds are reacted
with HSFM, or fragments thereof, and washed. Bound HSFM is then
detected by methods well known in the art. Purified HSFM can also
be coated directly onto plates for use in the aforementioned drug
screening techniques. Alternatively, non-neutralizing antibodies
can be used to capture the peptide and immobilize it on a solid
support.
[0181] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding HSFM specifically compete with a test compound for binding
HSFM. In this manner, antibodies can be used to detect the presence
of any peptide which shares one or more antigenic determinants with
HSFM.
[0182] In additional embodiments, the nucleotide sequences which
encode HSFM may be used in any molecular biology techniques that
have yet to be developed, provided the new techniques rely on
properties of nucleotide sequences that are currently known,
including, but not limited to, such properties as the triplet
genetic code and specific base pair interactions.
[0183] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
[0184] I. cDNA Library Construction
[0185] LUNGNOT14
[0186] The LUNGNOT14 cDNA library was constructed using RNA
isolated from lung tissue removed from the left lower lobe of a
47-year-old Caucasian male during a segmental lung resection.
Pathology for the associated tumor tissue indicated a grade 4
adenocarcinoma, and the parenchyma showed calcified granuloma.
Patient history included benign hypertension, chronic obstructive
pulmonary disease, and alcohol abuse. Family history included type
II diabetes and acute myocardial infarction.
[0187] PROSTUT12
[0188] The PROSTUT12 cDNA library was constructed using RNA
isolated from prostate tumor tissue from a 65-year-old Caucasian
male during a radical prostatectomy. Pathology indicated an
adenocarcinoma (Gleason grade 2+2). Adenofibromatous hyperplasia
was also present. The patient presented with elevated prostate
specific antigen (PSA).
[0189] LUNGNOT14 and PROSTUT12
[0190] The frozen tissue was homogenized and lysed in guanidinium
isothiocyanate solution using a Brinkmann Homogenizer Polytron
PT-3000 (Brinkmann Instruments, Westbury, N.Y.). The lysates were
centrifuged over a 5.7 M CsCl cushion using an Beckman SW28 rotor
in a Beckman L8-70M Ultracentrifuge (Beckman Instruments) for 18
hours at 25,000 rpm at ambient temperature. The RNA was extracted
with acid phenol, precipitated using sodium acetate and ethanol,
resuspended in RNAse-free water, and treated with DNase. The RNA
was extracted with acid phenol and precipitated as before. Poly(A+)
RNA was isolated using the Qiagen Oligotex kit (QIAGEN, Inc.,
Chatsworth, Calif.).
[0191] Poly(A+) RNA was used for cDNA synthesis and library
construction according to the recommended protocols in the
SuperScript.TM. plasmid system (Life Technologies, Inc.,
Gaithersburg, Md.). cDNAs were fractionated on a Sepharose CL4B
column (Pharmacia) and those cDNAs exceeding 400 bp were ligated
into the pINCY (Incyte Pharmaceuticals, Inc., Palo Alto, Calif.)
cloning vector and subsequently transformed into DH5.alpha..TM.
competent cells (Cat. #18258-012, Life Technologies, Inc.).
[0192] II. Isolation of cDNA Clones
[0193] Plasmid DNA was released from the cells and purified using
the REAL Prep 96 plasmid kit (QIAGEN, Inc.). The recommended
protocols were employed except for the following changes: I) the
bacteria were cultured in 1 ml of sterile Terrific Broth (Life
Technologies, Inc.) with carbenicillin at 25 mg/L and glycerol at
0.4%; 2) after the cultures were incubated for 19 hours, the cells
were lysed with 0.3 ml of lysis buffer; and 3) following
isopropanol precipitation, the plasmid DNA pellets were resuspended
in 0.1 ml of distilled water. The DNA samples were stored at
4.degree. C.
[0194] The cDNAs were sequenced by the method of Sanger et al.
(1975, J. Mol. Biol. 94:441f), using a Hamilton Micro Lab 2200
(Hamilton, Reno, Nev.) in combination with Peltier Thermal Cyclers
(PTC200 from MJ Research, Watertown, Mass.) and Applied Biosystems
377 DNA Sequencing Systems, and the reading frame was
determined.
[0195] III. Similarity Searching of cDNA Clones and Their Deduced
Proteins
[0196] The nucleotide sequences and/or amino acid sequences of the
Sequence Listing were used to query sequences in the GenBank,
SwissProt, BLOCKS, and Pima 11 databases. These databases, which
contain previously identified and annotated sequences, were
searched for regions of similarity using BLAST (Basic Local
Alignment Search Tool). (See, e.g., Altschul, S. F. (1993) J. Mol.
Evol 36:290-300; and Altschul et al., (1990) J. Mol. Biol.
215:403-410.)
[0197] BLAST produced alignments of both nucleotide and amino acid
sequences to determine sequence similarity. Because of the local
nature of the alignments, BLAST was especially useful in
determining exact matches or in identifying homologs which may be
of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant)
origin. Other algorithms could have been used when dealing with
primary sequence patterns and secondary structure gap penalties.
(See, e.g., Smith, T. et al. (1992) Protein Engineering 5:35-51.)
The sequences disclosed in this application have lengths of at
least 49 nucleotides and have no more than 12% uncalled bases
(where N is recorded rather than A, C, G, or T).
[0198] The BLAST approach searched for matches between a query
sequence and a database sequence. BLAST evaluated the statistical
significance of any matches found, and reported only those matches
that satisfy the user-selected threshold of significance. In this
application, threshold was set at 10.sup.-25 for nucleotides and
10.sup.-8 for peptides.
[0199] Incyte nucleotide sequences were searched against the
GenBank databases for primate (pri), rodent (rod), and other
mammalian sequences (mam), and deduced amino acid sequences from
the same clones were then searched against GenBank functional
protein databases, mammalian (mamp), vertebrate (vrtp), and
eukaryote (eukp), for similarity.
[0200] Additionally, sequences identified from cDNA libraries may
be analyzed to identify those gene sequences encoding conserved
protein motifs using an appropriate analysis program, e.g., BLOCKS.
BLOCKS is a weighted matrix analysis algorithm based on short amino
acid segments, or blocks, compiled from the PROSITE database.
(Bairoch, A. et al. (1997) Nucleic Acids Res. 25:217-221.) The
BLOCKS algorithm is useful for classifying genes with unknown
functions. (Henikoff, S. and Henikoff, G. J., Nucleic Acids
Research (1991) 19:6565-6572.) Blocks, which are 3 to 60 amino
acids in length, correspond to the most highly conserved regions of
proteins. The BLOCKS algorithm compares a query sequence with a
weighted scoring matrix of blocks in the BLOCKS database. Blocks in
the BLOCKS database are calibrated against protein sequences with
known functions from the SWISS-PROT database to determine the
stochastic distribution of matches. Similar databases such as
PRINTS, a protein fingerprint database, are also searchable using
the BLOCKS algorithm. (Attwood, T. K. et al. (1997) J. Chem. Inf.
Comput. Sci. 37:417-424.) PRINTS is based on non-redundant
sequences obtained from sources such as SWISS-PROT, GenBank, PIR,
and NRL-3D.
[0201] The BLOCKS algorithm searches for matches between a query
sequence and the BLOCKS or PRINTS database and evaluates the
statistical significance of any matches found. Matches from a
BLOCKS or PRINTS search can be evaluated on two levels, local
similarity and global similarity. The degree of local similarity is
measured by scores, and the extent of global similarity is measured
by score ranking and probability values. A score of 1000 or greater
for a BLOCKS match of highest ranking indicates that the match
falls within the 0.5 percentile level of false positives when the
matched block is calibrated against SWISS-PROT. Likewise, a
probability value of less than 1.0.times.10.sup.-3 indicates that
the match would occur by chance no more than one time in every 1000
searches. Only those matches with a cutoff score of 1000 or greater
and a cutoff probability value of 1.0.times.10.sup.-3 or less are
considered in the functional analyses of the protein sequences in
the Sequence Listing.
[0202] Nucleic and amino acid sequences of the Sequence Listing may
also be analyzed using PFAM. PFAM is a Hidden Markov Model (HMM)
based protocol useful in protein family searching. HMM is a
probabilistic approach which analyzes consensus primary structures
of gene families. (See, e.g., Eddy, S. R. (1996) Cur. Opin. Str.
Biol. 6:361-365.)
[0203] The PFAM database contains protein sequences of 527 protein
families gathered from publicly available sources, e.g., SWISS-PROT
and PROSITE. PFAM searches for well characterized protein domain
families using two high-quality alignment routines, seed alignment
and full alignment. (See, e.g., Sonnhammer, E. L. L. et al. (1997)
Proteins 28:405-420.) The seed alignment utilizes the hmmis
program, a program that searches for local matches, and a
non-redundant set of the PFAM database. The full alignment utilizes
the hmmfs program, a program that searches for multiple fragments
in long sequences, e.g., repeats and motifs, and all sequences in
the PFAM database. A result or score of 100 "bits" can signify that
it is 2.sup.100-fold more likely that the sequence is a true match
to the model or comparison sequence. Cutoff scores which range from
10 to 50 bits are generally used for individual protein families
using the SWISS-PROT sequences as model or comparison
sequences.
[0204] Two other algorithms, SIGPEPT and TM, both based on the HMM
algorithm described above (see, e.g., Eddy, supra; and Sonnhammer,
supra), identify potential signal sequences and transmembrane
domains, respectively. SIGPEPT was created using protein sequences
having signal sequence annotations derived from SWISS-PROT. It
contains about 1413 non-redundant signal sequences ranging in
length from 14 to 36 amino acid residues. TM was created similarly
using transmembrane domain annotations. It contains about 453
non-redundant transmembrane sequences encompassing 1579
transmembrane domain segments. Suitable HMM models were constructed
using the above sequences and were refined with known SWISS-PROT
signal peptide sequences or transmembrane domain sequences until a
high correlation coefficient, a measurement of the correctness of
the analysis, was obtained. Using the protein sequences from the
SWISS-PROT database as a test set, a cutoff score of 11 bits, as
determined above, correlated with 91-94% true-positives and about
4.1% false-positives, yielding a correlation coefficient of about
0.87-0.90 for SIGPEPT. A score of 11 bits for TM will typically
give the following results: 75% true positives; 1.72% false
positives; and a correlation coefficient of 0.76. Each search
evaluates the statistical significance of any matches found and
reports only those matches that score at least 11 bits.
[0205] IV. Northern Analysis
[0206] Northern analysis is a laboratory technique used to detect
the presence of a transcript of a gene and involves the
hybridization of a labeled nucleotide sequence to a membrane on
which RNAs from a particular cell type or tissue have been bound.
(See, e.g., Sambrook, supra, ch. 7; and Ausubel, supra, ch. 4 and
16.)
[0207] Analogous computer techniques applying BLAST are used to
search for identical or related molecules in nucleotide databases
such as GenBank or LIFESEQ.TM. database (Incyte Pharmaceuticals).
This analysis is much faster than multiple membrane-based
hybridizations. In addition, the sensitivity of the computer search
can be modified to determine whether any particular match is
categorized as exact or similar.
[0208] The basis of the search is the product score, which is
defined as: 1 % sequence identity .times. % maximum BLAST score
100
[0209] The product score takes into account both the degree of
similarity between two sequences and the length of the sequence
match. For example, with a product score of 40, the match will be
exact within a 1% to 2% error, and, with a product score of 70, the
match will be exact. Similar molecules are usually identified by
selecting those which show product scores between 15 and 40,
although lower scores may identify related molecules.
[0210] The results of Northern analysis are reported as a list of
libraries in which the transcript encoding HSFM occurs. Abundance
and percent abundance are also reported. Abundance directly
reflects the number of times a particular transcript is represented
in a cDNA library, and percent abundance is abundance divided by
the total number of sequences examined in the cDNA library.
[0211] V. Extension of HSFM Encoding Polynucleotides
[0212] The nucleic acid sequences of Incyte Clones 1511003 and
1810320 were used to design oligonucleotide primers for extending
partial nucleotide sequences to full length. For each nucleic acid
sequence, one primer was synthesized to initiate extension of an
antisense polynucleotide, and the other was synthesized to initiate
extension of a sense polynucleotide. Primers were used to
facilitate the extension of the known sequence "outward" generating
amplicons containing new unknown nucleotide sequence for the region
of interest. The initial primers were designed from the cDNA using
OLIGO.TM. 4.06 (National Biosciences, Plymouth, Minn.), or another
appropriate program, to be about 22 to 30 nucleotides in length, to
have a GC content of about 50% or more, and to anneal to the target
sequence at temperatures of about 68.degree. C. to about 72.degree.
C. Any stretch of nucleotides which would result in hairpin
structures and primer-primer dimerizations was avoided.
[0213] Selected human cDNA libraries (GIBCO BRL) were used to
extend the sequence. If more than one extension is necessary or
desired, additional sets of primers are designed to further extend
the known region.
[0214] High fidelity amplification was obtained by following the
instructions for the XL-PCR.TM. kit (The Perkin-Elmer Corp.,
Norwalk, Conn.) and thoroughly mixing the enzyme and reaction mix.
PCR was performed using the PTC-200 thermal cycler (MJ Research,
Inc., Watertown, Mass.), beginning with 40 pmol of each primer and
the recommended concentrations of all other components of the kit,
with the following parameters:
1 Step 1 94.degree. C. for 1 min (initial denaturation) Step 2
65.degree. C. for 1 min Step 3 68.degree. C. for 6 min Step 4
94.degree. C. for 15 sec Step 5 65.degree. C. for 1 min Step 6
68.degree. C. for 7 min Step 7 Repeat steps 4 through 6 for an
additional 15 cycles Step 8 94.degree. C. for 15 sec Step 9
65.degree. C. for 1 min Step 10 68.degree. C. for 7:15 min Step 11
Repeat steps 8 through 10 for an additional 12 cycles Step 12
72.degree. C. for 8 min Step 13 4.degree. C. (and holding)
[0215] A 5 .mu.l to 10 .mu.l aliquot of the reaction mixture was
analyzed by electrophoresis on a low concentration (about 0.6% to
0.8%) agarose mini-gel to determine which reactions were successful
in extending the sequence. Bands thought to contain the largest
products were excised from the gel, purified using QIAQUICK.TM.
(QIAGEN Inc.), and trimmed of overhangs using Klenow enzyme to
facilitate religation and cloning.
[0216] After ethanol precipitation, the products were redissolved
in 13 .mu.l of ligation buffer, 1 .mu.l T4-DNA ligase (15 units)
and 1 .mu.l T4 polynucleotide kinase were added, and the mixture
was incubated at room temperature for 2 to 3 hours, or overnight at
16.degree. C. Competent E. coli cells (in 40 .mu.l of appropriate
media) were transformed with 3 .mu.l of ligation mixture and
cultured in 80 .mu.l of SOC medium. (See, e.g., Sambrook, supra,
Appendix A, p. 2.) After incubation for one hour at 37.degree. C.,
the E. coli mixture was plated on Luria Bertani (LB) agar (See,
e.g., Sambrook, supra, Appendix A, p. 1) containing carbenicillin
(2.times. carb). The following day, several colonies were randomly
picked from each plate and cultured in 150 .mu.l of liquid
LB/2.times. carb medium placed in an individual well of an
appropriate commercially-available sterile 96-well microtiter
plate. The following day, 5 .mu.l of each overnight culture was
transferred into a non-sterile 96-well plate and, after dilution
1:10 with water, 5 .mu.l from each sample was transferred into a
PCR array.
[0217] For PCR amplification, 18 .mu.l of concentrated PCR reaction
mix (3.3.times.) containing 4 units of rTth DNA polymerase, a
vector primer, and one or both of the gene specific primers used
for the extension reaction were added to each well. Amplification
was performed using the following conditions:
2 Step 1 94.degree. C. for 60 sec Step 2 94.degree. C. for 20 sec
Step 3 55.degree. C. for 30 sec Step 4 72.degree. C. for 90 sec
Step 5 Repeat steps 2 through 4 for an additional 29 cycles Step 6
72.degree. C. for 180 sec Step 7 4.degree. C. (and holding)
[0218] Aliquots of the PCR reactions were run on agarose gels
together with molecular weight markers. The sizes of the PCR
products were compared to the original partial cDNAs, and
appropriate clones were selected, ligated into plasmid, and
sequenced.
[0219] In like manner, the nucleotide sequences of SEQ ID NO:3 and
SEQ ID NO:4 are used to obtain 5' regulatory sequences using the
procedure above, oligonucleotides designed for 5' extension, and an
appropriate genomic library.
[0220] VI. Labeling and Use of Individual Hybridization Probes
[0221] Hybridization probes derived from SEQ ID NO:3 and SEQ ID
NO:4 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although
the labeling of oligonucleotides, consisting of about 20 base
pairs, is specifically described, essentially the same procedure is
used with larger nucleotide fragments. Oligonucleotides are
designed using state-of-the-art software such as OLIGO.TM. 4.06
software (National Biosciences) and labeled by combining 50 .mu.mol
of each oligomer, 250 .mu.Ci of [.gamma.-.sup.32P] adenosine
triphosphate (Amersham, Chicago, Ill.), and T4 polynucleotide
kinase (DuPont NEN.RTM., Boston, Mass.). The labeled
oligonucleotides are substantially purified using a Sephadex.TM.
G-25 superfine size exclusion dextran bead column (Pharmacia &
Upjohn, Kalamazoo, Mich.). An aliquot containing 10.sup.7 counts
per minute of the labeled probe is used in a typical membrane-based
hybridization analysis of human genomic DNA digested with one of
the following endonucleases: Ase I, Bgl I, Eco RI, Pst I, XbaI, or
Pvu II (DuPont NEN, Boston, Mass.).
[0222] The DNA from each digest is fractionated on a 0.7% agarose
gel and transferred to nylon membranes (Nytran Plus, Schleicher
& Schuell, Durham, N.H.). Hybridization is carried out for 16
hours at 40.degree. C. To remove nonspecific signals, blots are
sequentially washed at room temperature under increasingly
stringent conditions up to 0.1.times. saline sodium citrate and
0.5% sodium dodecyl sulfate. After XOMAT AR.TM. film (Kodak,
Rochester, N.Y.) is exposed to the blots to film for several hours,
hybridization patterns are compared visually.
[0223] VII. Microarrays
[0224] A chemical coupling procedure and an ink jet device can be
used to synthesize array elements on the surface of a substrate.
(See, e.g., Baldeschweiler, supra.) An array analogous to a dot or
slot blot may also be used to arrange and link elements to the
surface of a substrate using thermal, UV, chemical, or mechanical
bonding procedures. A typical array may be produced by hand or
using available methods and machines and contain any appropriate
number of elements. After hybridization, nonhybridized probes are
removed and a scanner used to determine the levels and patterns of
fluorescence. The degree of complementarity and the relative
abundance of each probe which hybridizes to an element on the
microarray may be assessed through analysis of the scanned
images.
[0225] Full-length cDNAs, Expressed Sequence Tags (ESTs), or
fragments thereof may comprise the elements of the microarray.
Fragments suitable for hybridization can be selected using software
well known in the art such as LASERGENE.TM.. Full-length cDNAs,
ESTs, or fragments thereof corresponding to one of the nucleotide
sequences of the present invention, or selected at random from a
cDNA library relevant to the present invention, are arranged on an
appropriate substrate, e.g., a glass slide. The cDNA is fixed to
the slide using, e.g., UV cross-linking followed by thermal and
chemical treatments and subsequent drying. (See, e.g., Schena, M.
et al. (1995) Science 270:467-470, and Shalon, D. et al. (1996)
Genome Res. 6:639-645.) Fluorescent probes are prepared and used
for hybridization to the elements on the substrate. The substrate
is analyzed by procedures described above.
[0226] VIII. Complementary Polynucleotides
[0227] Sequences complementary to the HSFM-encoding sequences, or
any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring HSFM. Although use of
oligonucleotides comprising from about 15 to 30 base pairs is
described, essentially the same procedure is used with smaller or
with larger sequence fragments. Appropriate oligonucleotides are
designed using OLIGO.TM. 4.06 software and the coding sequence of
HSFM. To inhibit transcription, a complementary oligonucleotide is
designed from the most unique 5' sequence and used to prevent
promoter binding to the coding sequence. To inhibit translation, a
complementary oligonucleotide is designed to prevent ribosomal
binding to the HSFM-encoding transcript.
[0228] IX. Expression of HSFM
[0229] Expression and purification of HSFM is achieved using
bacterial or virus-based expression systems. For expression of HSFM
in bacteria, cDNA is subcloned into an appropriate vector
containing an antibiotic resistance gene and an inducible promoter
that directs high levels of cDNA transcription. Examples of such
promoters include, but are not limited to, the trp-lac (tac) hybrid
promoter and the T5 or T7 bacteriophage promoter in conjunction
with the lac operator regulatory element. Recombinant vectors are
transformed into suitable bacterial hosts, e.g., BL21 (DE3).
Antibiotic resistant bacteria express HSFM upon induction with
isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of HSFM
in eukaryotic cells is achieved by infecting insect or mammalian
cell lines with recombinant Autographica californica nuclear
polyhedrosis virus (AcMNPV), commonly known as baculovirus. The
nonessential polyhedrin gene of baculovirus is replaced with cDNA
encoding HSFM by either homologous recombination or
bacterial-mediated transposition involving transfer plasmid
intermediates. Viral infectivity is maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription.
Recombinant baculovirus is used to infect Spodoptera frugiperda
(Sf9) insect cells in most cases, or human hepatocytes, in some
cases. Infection of the latter requires additional genetic
modifications to baculovirus. (See Engelhard, E. K. et al. (1994)
Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig. V. et al. (1996)
Hum. Gene Ther. 7:1937-1945.)
[0230] In most expression systems, HSFM is synthesized as a fusion
protein with, e.g., glutathione S-transferase (GST) or a peptide
epitope tag, such as FLAG or 6-His, permitting rapid, single-step,
affinity-based purification of recombinant fusion protein from
crude cell lysates. GST, a 26-kilodalton enzyme from Schistosoma
japonicum, enables the purification of fusion proteins on
immobilized glutathione under conditions that maintain protein
activity and antigenicity (Pharmacia, Piscataway, N.J.). Following
purification, the GST moiety can be proteolytically cleaved from
HSFM at specifically engineered sites. FLAG, an 8-amino acid
peptide, enables immunoaffinity purification using commercially
available monoclonal and polyclonal anti-FLAG antibodies (Eastman
Kodak, Rochester, N.Y.). 6-His, a stretch of six consecutive
histidine residues, enables purification on metal-chelate resins
(QIAGEN Inc, Chatsworth, Calif.). Methods for protein expression
and purification are discussed in Ausubel, F. M. et al. (1995 and
periodic supplements) Current Protocols in Molecular Biology, John
Wiley & Sons, New York, N.Y., ch 10, 16. Purified HSFM obtained
by these methods can be used directly in the following activity
assay.
[0231] X. Demonstration of HSFM Activity
[0232] HSFM activity is demonstrated by the ability to oxidize
NADPH to NADP in the presence of substrate. (Kunau and Dommes
(1978) Eur. J. Biochem. 91:533-544.) Substrates include, but are
not limited to, all-trans-retinaldehyde and cis-4-dienoyl-CoA. HSFM
is preincubated for 10 min. at 37.degree. C. in 60 .mu.M potassium
phosphate (pH 7.4), 125 nM NADPH, and 0.2 .mu.M CoASH. The reaction
is started by addition of the appropriate substrate (12.5 to 150
.mu.M final concentration). Change in absorbance at 340 nm, due to
the oxidation of NADPH to NADP, is measured using a
spectrophotometer at 23.degree. C. Units of HSFM activity are
expressed as pmoles of NADP formed per minute. A reaction lacking
HSFM is used as a negative control.
[0233] XI. Functional Assays
[0234] HSFM function is assessed by expressing the sequences
encoding HSFM at physiologically elevated levels in mammalian cell
culture systems. cDNA is subcloned into a mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice include pCMV SPORT.TM. (Life
Technologies, Gaithersburg, Md.) and pCRT.TM. 3.1 (Invitrogen,
Carlsbad, Calif., both of which contain the cytomegalovirus
promoter. 5-10 .mu.g of recombinant vector are transiently
transfected into a human cell line, preferably of endothelial or
hematopoietic origin, using either liposome formulations or
electroporation. 1-2 .mu.g of an additional plasmid containing
sequences encoding a marker protein are co-transfected. Expression
of a marker protein provides a means to distinguish transfected
cells from nontransfected cells and is a reliable predictor of cDNA
expression from the recombinant vector. Marker proteins of choice
include, e.g., Green Fluorescent Protein (GFP) (Clontech, Palo
Alto, Calif.), CD64, or a CD64-GFP fusion protein. Flow cytometry
(FCM), an automated, laser optics-based technique, is used to
identify transfected cells expressing GFP or CD64-GFP, and to
evaluate properties, for example, their apoptotic state. FCM
detects and quantifies the uptake of fluorescent molecules that
diagnose events preceding or coincident with cell death. These
events include changes in nuclear DNA content as measured by
staining of DNA with propidium iodide; changes in cell size and
granularity as measured by forward light scatter and 90 degree side
light scatter; down-regulation of DNA synthesis as measured by
decrease in bromodeoxyuridine uptake; alterations in expression of
cell surface and intracellular proteins as measured by reactivity
with specific antibodies; and alterations in plasma membrane
composition as measured by the binding of fluorescein-conjugated
Annexin V protein to the cell surface. Methods in flow cytometry
are discussed in Ormerod, M. G. (1994) Flow Cytometry, Oxford, New
York, N.Y.
[0235] The influence of HSFM on gene expression can be assessed
using highly purified populations of cells transfected with
sequences encoding HSFM and either CD64 or CD64-GFP. CD64 and
CD64-GFP are expressed on the surface of transfected cells and bind
to conserved regions of human immunoglobulin G (IgG). Transfected
cells are efficiently separated from nontransfected cells using
magnetic beads coated with either human IgG or antibody against
CD64 (DYNAL, Lake Success, N.Y.). mRNA can be purified from the
cells using methods well known by those of skill in the art.
Expression of mRNA encoding HSFM and other genes of interest can be
analyzed by Northern analysis or microarray techniques.
[0236] XII. Production of HSFM Specific Antibodies
[0237] HSFM substantially purified using polyacrylamide gel
electrophoresis (PAGE)(see, e.g., Harrington, M. G. (1990) Methods
Enzymol. 182:488495), or other purification techniques, is used to
immunize rabbits and to produce antibodies using standard
protocols.
[0238] Alternatively, the HSFM amino acid sequence is analyzed
using LASERGENE.TM. software (DNASTAR Inc.) to determine regions of
high immunogenicity, and a corresponding oligopeptide is
synthesized and used to raise antibodies by means known to those of
skill in the art. Methods for selection of appropriate epitopes,
such as those near the C-terminus or in hydrophilic regions are
well described in the art. (See, e.g., Ausubel supra, ch. 11.)
[0239] Typically, oligopeptides 15 residues in length are
synthesized using an Applied Biosystems Peptide Synthesizer Model
431A using fmoc-chemistry and coupled to KLH (Sigma, St. Louis,
Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester
(MBS) to increase immunogenicity. (See, e.g., Ausubel supra.)
Rabbits are immunized with the oligopeptide-KLH complex in complete
Freund's adjuvant. Resulting antisera are tested for antipeptide
activity by, for example, binding the peptide to plastic, blocking
with 1% BSA, reacting with rabbit antisera, washing, and reacting
with radio-iodinated goat anti-rabbit IgG.
[0240] XIII. Purification of Naturally Occurring HSFM Using
Specific Antibodies
[0241] Naturally occurring or recombinant HSFM is substantially
purified by immunoaffinity chromatography using antibodies specific
for HSFM. An immunoaffinity column is constructed by covalently
coupling anti-HSFM antibody to an activated chromatographic resin,
such as CNBr-activated Sepharose (Pharmacia & Upjohn). After
the coupling, the resin is blocked and washed according to the
manufacturer's instructions.
[0242] Media containing HSFM are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of HSFM (e.g., high ionic strength buffers
in the presence of detergent). The column is eluted under
conditions that disrupt antibody/HSFM binding (e.g., a buffer of pH
2 to pH 3, or a high concentration of a chaotrope, such as urea or
thiocyanate ion), and HSFM is collected.
[0243] XIV. Identification of Molecules which Interact with
HSFM
[0244] HSFM, or biologically active fragments thereof, are labeled
with .sup.125I Bolton-Hunter reagent. (See, e.g., Bolton et al.
(1973) Biochem. J. 133:529.) Candidate molecules previously arrayed
in the wells of a multi-well plate are incubated with the labeled
HSFM, washed, and any wells with labeled HSFM complex are assayed.
Data obtained using different concentrations of HSFM are used to
calculate values for the number, affinity, and association of HSFM
with the candidate molecules.
[0245] Various modifications and variations of the described
methods and systems of the invention will be apparent to those
skilled in the art without departing from the scope and spirit of
the invention. Although the invention has been described in
connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly
limited to such specific embodiments. Indeed, various modifications
of the described modes for carrying out the invention which are
obvious to those skilled in molecular biology or related fields are
intended to be within the scope of the following claims.
Sequence CWU 1
1
* * * * *