U.S. patent application number 10/690019 was filed with the patent office on 2004-05-27 for fibroblast growth factor homologous factors (fhfs) and methods of use.
This patent application is currently assigned to THE JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE. Invention is credited to Nathans, Jeremy, Smallwood, Philip M..
Application Number | 20040102379 10/690019 |
Document ID | / |
Family ID | 24832639 |
Filed Date | 2004-05-27 |
United States Patent
Application |
20040102379 |
Kind Code |
A1 |
Nathans, Jeremy ; et
al. |
May 27, 2004 |
Fibroblast growth factor homologous factors (FHFs) and methods of
use
Abstract
The invention provides fibroblast growth factor homologous
factor (FHF) polypeptides and nucleic acid molecules that encode
them. Also included in the invention are diagnostic and therapeutic
methods using FHF polypeptides and nucleic acids.
Inventors: |
Nathans, Jeremy; (Baltimore,
MD) ; Smallwood, Philip M.; (Woodbine, MD) |
Correspondence
Address: |
Lisa A. Haile, J.D., Ph.D.
GRAY CARY WARE & FREIDENRICH LLP
4365 Executive Drive, Suite 1100
San Diego
CA
92121-2133
US
|
Assignee: |
THE JOHNS HOPKINS UNIVERSITY SCHOOL
OF MEDICINE
|
Family ID: |
24832639 |
Appl. No.: |
10/690019 |
Filed: |
October 20, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10690019 |
Oct 20, 2003 |
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09490714 |
Jan 25, 2000 |
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6635744 |
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09490714 |
Jan 25, 2000 |
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08705245 |
Aug 30, 1996 |
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6020189 |
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Current U.S.
Class: |
435/6.16 ;
435/320.1; 435/325; 435/69.1; 514/9.1; 530/399; 536/23.5 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 37/00 20180101; A61P 43/00 20180101; A61P 27/02 20180101; Y10S
530/814 20130101; A61K 38/00 20130101; A61P 25/16 20180101; C07K
14/50 20130101; Y10S 530/811 20130101; Y10S 530/827 20130101; A61P
25/02 20180101; A61P 25/28 20180101; Y10S 530/81 20130101; A61P
31/12 20180101; Y10S 530/815 20130101; Y10S 530/806 20130101; Y10S
530/812 20130101; A61P 15/08 20180101; Y10S 530/813 20130101 |
Class at
Publication: |
514/012 ;
435/069.1; 435/320.1; 435/325; 530/399; 536/023.5 |
International
Class: |
C07K 014/50; C12Q
001/68; A61K 038/18 |
Claims
1. A substantially pure fibroblast growth factor homologous factor
(FHF) polypeptide.
2. The polypeptide of claim 1, wherein the polypeptide: a. is about
225-250 amino acids in length; b. lacks an amino terminal signal
sequence; and c. contains a nuclear localization signal.
3. The polypeptide of claim 1, wherein the polypeptide comprises a
segment of at least five consecutive amino acids that are conserved
in the amino acid sequences of FHF-1 (SEQ ID NO:1), FHF-2 (SEQ ID
NO:2), FHF-3 (SEQ ID NO:3), and FHF-4 (SEQ ID NO:4).
4. The polypeptide of claim 3, wherein the segment comprises at
least five amino acids of the sequence of SEQ ID NO:22.
5. The polypeptide of claim 3, wherein the segment comprises at
least five amino acids of the sequence of SEQ ID NO:24.
6. The polypeptide of claim 1, wherein the polypeptide is
FHF-4.
7. The polypeptide of claim 1, wherein the polypeptide comprises
the amino acid sequence of FIG. 8 (SEQ ID NO:4).
8. An isolated nucleic acid encoding the FHF polypeptide of claim
1.
9. The nucleic acid of claim 8, wherein the sequence of the nucleic
acid comprises the nucleotide sequence of FIG. 8 (SEQ ID
NO:21).
10. The nucleic acid of claim 8, wherein the sequence of the
nucleic acid is selected from the group consisting of: a. the
nucleotide sequence of FIG. 8 (SEQ ID NO:21), where T can also be
U; b. a nucleic acid sequence that hybridizes to the complement of
the nucleotide sequence of FIG. 8 (SEQ ID NO:21); and c: a fragment
of a. or b. that comprises at least 15 nucleotides and hybridizes
to the complement of the nucleotide sequence of FIG. 8 (SEQ ID
NO:21).
11. A nucleic acid that hybridizes to the nucleic acid of claim
8.
12. A nucleic acid that hybridizes to the nucleic acid of claim
10.
13. The nucleic acid of claim 8, wherein the nucleic acid is
mammalian.
14. The nucleic acid of claim 13, wherein the nucleic acid is
human.
15. An expression vector containing the nucleic acid of claim
8.
16. The vector of claim 15, wherein the vector is a plasmid.
17. The vector of claim 15, wherein the vector is a virus.
18. A cell stably transformed with the vector of claim 15.
19. An antibody that binds to the FHF polypeptide of claim 1.
20. The antibody of claim 19, wherein the antibody is
monoclonal.
21. A method of detecting a cell proliferative disorder associated
with expression of the FHF polypeptide of claim 1, the method
comprising the steps of: a. contacting a specimen from a subject
having or suspected of having the disorder with a reagent that
detects expression of the FHF polypeptide, and b. detecting binding
of the reagent to the specimen.
22. The method of claim 21, wherein the cell is a brain cell.
23. The method of claim 21, wherein the reagent is an antibody.
24. The method of claim 21, wherein the reagent is a nucleic
acid.
25. The method of claim 24, wherein the nucleic acid hybridizes to
the nucleic acid of claim 8.
26. The method of claim 24, wherein the nucleic acid hybridizes to
the complement of the nucleic acid of claim 8.
27. The method of claim 21, wherein the detecting is carried out in
vivo.
28. The method of claim 21, wherein the detecting is carried out in
vitro.
29. The method of claim 21, wherein the reagent comprises a
detectable label.
30. A method of treating a cell proliferative disorder associated
with expression of the FHF polypeptide of claim 1, the method
comprising administering to a subject having or suspected of having
the disorder a reagent that suppresses the activity of the FHF
polypeptide.
31. The method of claim 30, wherein the reagent is an anti-FHF
antibody.
32. The method of claim 30, wherein the reagent is a nucleic acid
that hybridizes to the nucleic acid of claim 8.
33. The method of claim 30, wherein the cell is a brain cell.
34. The method of claim 30, wherein the reagent is introduced into
the cell using a carrier.
35. The method of claim 34, wherein the carrier is a vector.
36. A method of identifying a nucleic acid encoding an FHF
polypeptide, the method comprising probing a sample containing a
nucleic acid encoding an FHF polypeptide with an FHF-specific
nucleic acid probe.
37. The method of claim 36, wherein the FHF-specific nucleic acid
probe hybridizes to: a. a nucleic acid that encodes seven
consecutive amino acids, at least four of which are conserved in
the amino acid sequences of FHF-1 (SEQ ID NO:1), FHF-2 (SEQ ID
NO:2), FHF-3 (SEQ ID NO:3), and FHF-4 (SEQ ID NO:4), or b. the
complementary sequence thereto.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The invention relates generally to polypeptide growth
factors and specifically to fibroblast growth factor homologous
factors (FHFs) and nucleic acids encoding FHFs.
[0003] 2. Description of Related Art
[0004] The fibroblast growth factor (FGF) family encompasses a
group of structurally related proteins with a wide range of growth
promoting, survival, and differentiation activities in vivo and in
vitro (reviewed in Baird and Gospodarowicz, N.Y. Acad. Sci., 638:1,
1991; Eckenstein, J. Neurobiology, 25:1467, 1994; Mason, Cell,
78:547, 1994). As of June, 1996, nine members of this family had
been characterized by molecular cloning and their sequences
published. The first two members of the-family to be characterized,
acidic FGF (aFGF/FGF-1) and basic FGF (bFGF/FGF-2), have been found
in numerous tissues, including brain, eye, kidney, placenta, and
adrenal tissues (Jaye, et al., Science, 233:541, 1986; Abraham, et
al., Science, 233:545, 1986). These factors have been shown to be
potent mitogens and survival factors for a variety of mesoderm and
neuroectoderm-derived tissues, including fibroblasts, endothelial
cells, hippocampal and cerebral cortical neurons, and astroglia
(Burgess and Maciag, Ann. Rev. Biochemistry, 58:575, 1989). Another
member of the FGF family is int-2/FGF-3, which is encoded by a gene
that is a common target for activation by the mouse mammary tumor
virus, and therefore is presumed to be an oncogenic factor (Smith,
et al., EMBO J., 7:1013, 1988). The genes encoding FGF-4, FGF-5,
and FGF-6 have transforming activity when introduced into NIH 3T3
cells (Delli-Bovi, et al., Cell, 50:729, 1987; Zhan, et al., Mol.
Cell. Biol., 8:3487, 1988; Marics, et al., Oncogene, 4:335, 1989),
while keratinocyte growth factor (KGF)/FGF-7, FGF-8, and FGF-9 are
mitogenic for keratinocytes, mammary carcinoma cells, and
astrocytes, respectively (Finch, et al., Science, 245:752, 1989;
Tanaka, et al., Proc. Natl. Acad. Sci. USA, 89:8928, 1992;
Miyamoto, et al., Mol. Cell Biol., 13:4251, 1993). Recent
experiments indicate that several FGFs have bioactivities that were
not evident during their initial identification. For example, FGF-2
has been shown to induce ventral mesoderm in Xenopus embryos
(Slack,. et al., Nature 326:197-200, 1987; Kimmelman, et al., Cell
51:869-877, 1989), FGF-4 has been shown to be involved in growth
and patterning of the chick limb bud (Niswander, et al., Nature
371:609-612, 1994), FGF-5 has been shown to control hair follicle
cycling in the mouse (Hebert, Cell 78:1017-1025, 1994), and FGF-8
has been shown to cause duplications of the embryonic chick
midbrain (Crossley, et al, Nature 380:66-68, 1996). Several of the
FGFs, including aFGF (FGF-1) and bFGF (FGF-2), lack classical
signal sequences, and the mechanism by which they are secreted is
not known. Current data indicate that FGF-1 and FGF-2 are released
from cells by a route that is distinct from the ER-Golgi secretory
pathway (Florkiewicz, et al., J. Cell Physiol. 162:388-399, 1995;
Jackson, et al., J. Biol. Chem. 270:33-36, 1995).
[0005] The nine published members of the FGF family, FGFs 1-9, are
between 155 and 268 amino acids in length and share approximately
25% or more amino acid sequence identity, as well as a conserved
central region of approximately 140 amino acids. This region forms
a compact beta-barrel with three-fold symmetry that is nearly
identical in structure to the folded core of interleukins 1-alpha
and 1-beta (Zhu, et al., Science 251:90-93, 1991; Zhang, et al.,
Proc. Natl Acad. Sci. USA 88:3446-3450,1991; Eriksson, et al.,
Proc. Natl Acad. Sci. USA 88:3441-3445, 1991; Ago, et al., J.
Biochem. 110:360-363, 1991). FGF-1 and FGF-2 also resemble
interleukin 1-beta in lacking a classical signal sequence.
[0006] FGF signaling is generally thought to occur by activation of
transmembrane tyrosine kinase receptors. For example, FGF-1, FGF-2,
and FGF-7/KGF have been shown to exert some or all of their
biological activities through high affinity binding to such
receptors (see, e.g., Lee, et al., Science, 245:57, 1989; reviewed
in Johnson and Williams, Adv. Cancer Res., 60:1, 1993). Four FGF
receptor (FGFR) genes have been identified thus far (Johnson, et
al., Adv. Cancer Res. 60:1-41, 1993), and activating or
inactivating receptor mutations have been described for a subset of
these genes, in both mice and humans. In the mouse, disruption of
the FGFR1 or FGFR2 genes leads to early embryonic lethality (Deng,
et al., Genes Dev. 8:3045-3057, 1994; Yamaguchi, et al., Genes Dev.
8:3032-3044, 1994), and disruption of FGFR3 leads to bone
overgrowth (Deng, et al., Cell 84:911-921, 1996; Colvin, et al.,
Nature Genet. 12:390-397, 1996). In humans, point mutations in
FGFR1, FGFR2, and FGFR3 have been found in a variety of skeletal
disorders (reviewed by Muenke and Schell, Trends Genet. 11,
308-313, 1995). Recent work has shown that receptor diversity is
increased by alternative pre-mRNA splicing within the extracellular
ligand binding domain, with the result that multiple receptor
isoforms, with different ligand binding properties, can be encoded
by the same gene (Johnson and Williams, supra). In tissue culture
systems, binding of aFGF or bFGF to its cell surface receptor
activates phospholipase C-gamnna (Burgess, et al., Mol. Cell Biol.,
10:4770, 1990), which is a component of a pathway known to
integrate a variety of mitogenic signals. Many members of the FGF
family also bind tightly to heparin, and a ternary complex of
heparin, FGF, and a transmembrane receptor may be a biologically
relevant signaling species.
SUMMARY OF THE INVENTION
[0007] The invention provides fibroblast growth factor homologous
factor (FHF) polypeptides and nucleic acids that encode them. FHFs
are involved in regulating the growth, survival, and
differentiation of cells in the central nervous system (CNS), as
well as cells in peripheral nervous tissues.
[0008] The invention also provides methods for detecting
alterations in FHF gene expression, which can be used in the
diagnosis of neurodegenerative and neoplastic disorders. Methods
for treating neurodegenerative and neoplastic disorders, in which
the expression and/or activity of an FHF is modulated, are also
included in the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 shows an alignment o the amino acid sequences of
FHF-1 (SEQ ID NO: 1), FHF-2 (SEQ ID NO:2), FHF-3 (SEQ ID NO:3), and
FHF4 (SEQ ID NO:4). Amino acids that are conserved in all four of
FHFs 1-4 are shaded and boxed and amino acids that are conserved in
only some of FHFs 1-4 are shaded.
[0010] FIG. 2 shows an alignment of the amino acid sequences of
FHF-1 (SEQ ID NO: 1), FHF-2 (SEQ ID NO:2), FHF-3 (SEQ ID NO:3),
FHF-4 (SEQ ID NO:4), FGF-1 (SEQ ID NO:5), FGF-2 (SEQ ID NO:6), and
FGF-9 (SEQ ID NO:7). The large, black dots indicate amino acids
that are identical among FHFs 1-4, but that are different in the
nine previously characterized FGFs (FGFs 1-9). Intron locations for
murine FHF-2 are indicated by arrowheads above the aligned
sequences. The locations of the twelve segments having beta-sheet
conformations in the FGF-2 crystal structure are underlined
(Erickson, et al., Proc. Natl. Acad. Sci. USA 88:3441-3445,
1991).
[0011] FIG. 3 shows an alignment of the amino acid sequences of
mouse and human FHFs with each of the nine, previously
characterized members of the FGF family (hFHF-1 (SEQ ID NO: 1),
hFHF-2 (SEQ ID NO:2), hFHF-3 (SEQ ID NO:3), hFHF-4 (SEQ ID NO:4),
mFHF-1 (SEQ ID NO:8), mFHF-2 (SEQ ID NO:9), mFHF-3 (SEQ ID NO:10),
and mnFHF-4 (SEQ ID NO:11)).
[0012] The FGF family members include aFGF/FGF-1 (SEQ ID NO:5;
Jaye, et al., supra), bFGF/FGF-2 (SEQ ID NO:6; Abraham, et al.,
supra), int-2/FGF-3 (SEQ ID NO:12; Smith, et al., supra), FGF-4
(SEQ ID NO: 13; Delli-Bovi, et al., supra), FGF-5 (SEQ ID NO: 14;
Zhan, et al., supra), FGF-6 (SEQ ID NO: 15; Maricas, et al.,
supra), keratinocyte growth factor/FGF-7 (SEQ ID NO:16; Finch, et
al., supra), FGF-8 (SEQ ID NO:17; Tanaka, et al., supra), and FGF-9
(SEQ ID NO:7; Miyamoto, et al., supra). ("m" denotes mouse, and "h"
denotes human.)
[0013] FIG. 4 shows a dendrogram of mammalian FHF and FGF family
members, in which the length of each path connecting any pair of
FHF or FGF family members is proportional to the degree of amino
acid sequence divergence of that pair: ("m" denotes mouse, and "h"
denotes human.)
[0014] FIG. 5 shows the nucleotide (SEQ ID NO:18) and deduced amino
acid (SEQ ID NO:1) sequences of FHF-1.
[0015] FIG. 6 shows the nucleotide (SEQ ID NO: 19) and deduced
amino acid (SEQ ID NO:2) sequences of FHF-2.
[0016] FIG. 7 shows the nucleotide (SEQ ID NO:20) and deduced amino
acid (SEQ ID NO:3) sequences of FHF-3.
[0017] FIG. 8 shows the nucleotide (SEQ ID NO:21) and deduced amino
acid (SEQ ID NO:4) sequences of FHF4.
[0018] FIG. 9 shows partial chromosome linkage maps for mouse
FHF-1, FHF-2, and. FHF-3 genes. The genes were mapped by
interspecific backcross analysis. To the left of each chromosome
map, the number of recombinant N2 animals is presented, divided by
the total number of N2 animals typed for each pair of loci. The
recombination frequencies, expressed as genetic distance in
centimorgans (.+-. one standard error) are also shown. The upper
95% confidence limit of the recombination distance is given in
parentheses, in cases where no recombinants were found between
loci. The positions of loci on human chromosomes, where known, are
shown to the right of the chromosome maps. References for the map
positions of most human loci can be obtained from the GDB (Genome
Data Base), which is a computerized database of human linkage
information maintained by The William H. Welch Medical Library of
The Johns Hopkins University (Baltimore, Md.).
[0019] FIG. 10 shows the tissue distribution of FHF transcripts in
the adult mouse. Ten micrograms of total RNA from various mouse
tissues was prepared (Chomczinski and Sacchi, Anal. Biochem.,
162:156, 1987) and used in RNAse protection experiments (Ausubel,
et al., Current Protocols in Molecular Biology, Wiley Interscience,
New York, N.Y., 1987) employing the indicated antisense riboprobes
(FHFs 1-4). The RNA samples are as follows: 1, brain; 2, eye; 3,
heart; 4, kidney; 5, liver; 6, lung; 7, spleen; 8, testis; and 9,
yeast tRNA. A control reaction, in which an RNA Polymerase II probe
was used, is shown at the bottom of the figure.
[0020] FIG. 11 shows in situ localization of FHF transcripts in
sections prepared from the developing and adult mouse. .sup.33P in
situ hybridization is shown in red and is superimposed on a cresyl
violet stain shown in black and white. The probes and samples used
are as follows: (A) FHF-2, e11; (B) FHF-3, e11; (C, D) FHF-2, e17;
(E) FHF-1, P1, coronal section through the head at the level of the
eyes; (F) FHF-2, P1, coronal section through the center of the
head; (G) FHF-1, adult; (H, I) FHF-2, adult; (J) FHF-3, adult; (K,
L) FHF-4, adult.
[0021] FIG. 12 shows FHF-1 immunostaining in sections of the
macaque monkey CNS. The samples stained are as follows: (A)
precentral. motor cortex; (B) area 3b in the primary somatosensory
cortex; (C) primary auditory cortex; (D) area 7b in the superior
parietal lobule; and (E) primary visual cortex. Panels F-H show
that populations of cortical neurons immunoreactive for FHF-1
include large intensely, immunoreactive cells (arrows), small,
weakly immunoreactive cells (arrowheads), and small, weakly
immunoreactive cells, with fine processes resembling microglia
(double arrows). Simultaneous immunostaining for parvalbumin (G)
and FHF-1(H) shows that large and small neurons are immunoreactive
for both. An uneven distribution of FHF-1 immunoreactive somata is
seen in the hippocampal formation (I), including the subicular
complex (S), the CA fields, and the dentate gyrus (DG). In the
dorsal thalamus (J), FHF-1 immunoreactive somata occupy patches in
the caudal ventroposteriolateral nucleus (VPLc) and are found in
both magnocellular and parvicellular layers of the lateral
geniculate nucleus (LGN). The medial geniculate complex contains
few immunostained cells. In the basal telencephalon (K),
immunoreactive neurons are present in both the external and
internal segments of the globus pallidus (Gpe and GPi). Scale bars:
500 .mu.m in (A-E), 20 .mu.m in (F), 75 .mu.m in (G, H), and 1 mm
in (I-K).
[0022] FIG. 13 shows that FHF-1 is not secreted by 293 cells, which
are human embryonic kidney cells. 293 cells were transiently
transfected with plasmids directing expression of human growth
hormone (left 2 lanes; hGH), FHF-1I (center 2 lanes), or the human
red cone pigment (right 2 lanes; Red). The cells were labeled for 6
hours with .sup.35S-methionine in serum-free medium, and the total
protein present in the cells (C) or medium (M) was resolved by
SDS-PAGE and visualized by autoradiography. Secretion of hGH, but
not FHF-1, is observed. The mobilities of protein standards are
indicated at the left of the figure; from top to bottom, their
molecular masses, in kDa, are: 220, 97, 66, 46, 30, 21.5, and 14.3.
The mobilities of hGH and FHF-1 are indicated at the right side of
the figure.
[0023] FIG. 14 shows a summary of FHF-1 constructs used to identify
the FHF-1 nuclear localization signal (NLS). Localization of these
constructs by immunostaining (constructs 1-4) and localization of
FHF-1-.beta.-galactosidase fusions by X-gal staining (constructs
5-12) is also shown. The numbers underneath each construct indicate
the amino acids from FHF-1 that are present in the construct. (N,
nuclear staining; C, cytoplasmic staining; ++, strong staining; +,
weak staining; -, no staining. `a`, construct 12 shows cytoplasmic
staining in 15%-20% of cells and nuclear localization in 80%-85% of
cells.)
[0024] FIG. 15 shows double label immunofluorescent localization of
the constructs illustrated in FIG. 14. The antibodies used are as
follows: (A, B) double label immunofluorescent localization of
FHF-1 (green) and BiP (an ER marker; red), optically sectioned at
0.7 .mu.m; and (C-F) histochemical localization of
FHF-1-.beta.-galactosidase fusion proteins. All experiments were
performed in transiently transfected 293 cells. The constructs used
are: (A) full length FHF-1, construct 1; (B) construct 4; (C) full
length .beta.-galactosidase; (D) construct 6; (E) construct 11; (F)
construct 12.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The invention provides polypeptide growth factors,
designated fibroblast growth factor homologous factors (FHFs), and
nucleic acids that encode them. Genes encoding four FHFs,
designated FHFs 1-4, have been isolated and sequenced. The deduced
amino acid sequence of FHF-1 is 27% identical to that of FGF-9, and
the amino acid sequences of FHFs 1-4 are 58-70% identical to each
other. Thus, FHFs define a new branch of the FGF family.
[0026] FHFs are expressed in the developing and adult nervous
systems, and thus are believed to play roles in regulating nervous
system development and function. Accordingly, FHF polypeptides, and
nucleic acids that encode them, can be used in methods for treating
and diagnosing conditions affecting the nervous system, including,
e.g., stroke, neurodegenerative diseases, such as Parkinson's
disease and Alzheimer's disease, retinal degenerative diseases,
such as retinitis pigmentosa and macular degeneration, cerebellar
degenerative diseases, and cancer. More specific uses for
FHF-related molecules can be gleaned from their tissue
specificities. For example, although FHFs 1-4 are all expressed in
the brain, FHFs 1-3 are specifically expressed in the eye, FHFs 1
and 4 are expressed in the testes, and FHF-2 is expressed in the
heart. Thus, monoclonal and polyclonal antibodies can be produced
using standard immunization and screening methods well known in the
art. These antibodies can be easily detectably labelled and used
histologically to identify tissues which contain a given FHF. FHFs
can also be used in methods for maintaining cultured cells or
tissues, such as neuronal cells or tissues, prior to
transplantation. In addition, FHFs can be used to promote neuron
growth in vitro, in order to, for example, facilitate production of
growth factors, such as interleukin-2 (IL-2), that are produced by
them. Methods employing FHF polypeptides and nucleic acids are
described in further detail below.
[0027] The invention provides substantially pure FHF polypeptides.
FHF polypeptides can be characterized as containing, for example,
at least five consecutive amino acids that are conserved in at
least two, e.g., three or four, FHFs, such as FHFs 1-4. One or more
(e.g., two to four) of the five conserved amino acids, in addition
to being conserved in FHFs, can be characterized as not being
conserved in any of the nine previously characterized FGFs (FGFs
1-9, see above).
[0028] The term "substantially pure" is used herein to describe a
molecule, such as a polypeptide (e.g., an FHF polypeptide, or a
fragment thereof) that is substantially free of other proteins,
lipids, carbohydrates, nucleic acids, and other biological
materials with which it is naturally associated. For example, a
substantially pure molecule, such as a polypeptide, can be at least
60%, by dry weight, the molecule of interest. One skilled in the
art can purify FHF polypeptides using standard protein purification
methods and the purity of the polypeptides can be determined using
standard methods including, e.g., polyacrylamide gel
electrophoresis (e.g., SDS-PAGE), column chromatography (e.g., high
performance liquid chromatography (HPLC)), and amino-terminal amino
acid sequence analysis.
[0029] FHF polypeptides included in the invention can have one of
the amino acid sequences of FHFs 1-4, for example, the amino acid
sequence of FHF4. FHF polypeptides, such as FHFs 1-4, can be
characterized by being expressed in the brain, lacking classical
signal sequences, containing nuclear localization signals or
nuclear localization-like signals, and containing, at full length,
about 225-250 amino acids (FHF-1: 244 amino acids; FHF-2: 245 amino
acids; FHF-3: 225 amino acids; FHF4: 247 amino acids; see, e.g.,
FIGS. 1-3 and 5-8). The FHF polypeptides of the invention can be
derived from a mammal, such as a human or a mouse.
[0030] Also included in the invention are polypeptides having
sequences that are "substantially identical" to the sequence of an
FHF polypeptide, such as one of FHFs 1-4, e.g., FHF-4. A
"substantially identical" amino acid sequence is a sequence that
differs from a reference sequence only by conservative amino acid
substitutions, for example, substitutions of one amino acid for
another of the same class (e.g., substitution of one hydrophobic
amino acid,, such as isoleucine, valine, leucine, or methionine,
for another, or substitution of one polar amino acid for another,
such as substitution of arginine for lysine, glutamic acid for
aspartic acid, or glutamine for asparagine), or by one or more
non-conservative substitutions, deletions, or insertions, provided
that the polypeptide retains at least one FHF-specific activity or
an FHF-specific epitope. For example, one or more amino acids can
be deleted from an FHF polypeptide, resulting in modification of
the structure of the polypeptide, without significantly altering
its biological activity. For example, amino- or carboxyl-terminal
amino acids that are not required for FHF biological activity, can
be removed. Such modifications can result in the development of
smaller active FHF polypeptides.
[0031] Other FHF polypeptides included in the invention are
polypeptides having amino acid sequences that are at least 50%
identical to the amino acid sequence of an FHF polypeptide, such as
any of FHFs 1-4, e.g., FHF-4. The length of comparison in
determining amino acid sequence homology can be, for example, at
least 15 amino acids, for example, at least 20, 25, or 35 amino
acids. Homology can be measured using standard sequence analysis
software (e.g., Sequence Analysis Software Package of the Genetics
Computer Group, University of Wisconsin Biotechnology Center, 1710
University Avenue, Madison, Wis. 53705; also see Ausubel, et al.,
supra).
[0032] The invention also includes fragments of FHF polypeptides,
such as FHFs 1-4, that retain at least one FHF-specific activity or
epitope. For example, an FHF polypeptide fragment containing, e.g.,
at least 8-10 amino acids can be used as an immunogen in the
production of FHF-specific antibodies. The fragment can contain,
for example, an amino acid sequence that is conserved in FHFs, and
this amino acid sequence can contain amino acids that are conserved
in FHFs, but not in FGFs 1-9. Such fragments can easily be
identified by comparing the sequences of FHFs and FGFs, e.g., by
reference to FIGS. 1-3. In addition to their use as peptide
immunogens, the above-described FHF fragments can be used in
immunoassays, such as ELISAs, to detect the presence of
FHF-specific antibodies in samples.
[0033] The FHF polypeptides of the invention can be obtained using
any of several standard methods. For example, FHF polypeptides can
be produced in a standard recombinant expression systems (see
below), chemically synthesized (this approach may be limited to
small FHF peptide fragments), or purified from tissues in which
they are naturally expressed (see, e.g., Ausubel, et al.,
supra).
[0034] The invention also provides isolated nucleic acid molecules
that encode the FHF polypeptides described above, as well as
fragments thereof For example, nucleic acids that encode any of
FHFs 14, such as FHF4, are included in the invention. These nucleic
acids can contain naturally occurring nucleotide sequences (see
FIGS. 1-3 and 5-8), or sequences that differ from those of the
naturally occurring nucleic acids that encode FHFs 1-4, but encode
the same amino acids, due to the degeneracy of the genetic code.
The nucleic acids of the invention can contain DNA or RNA
nucleotides, or combinations or modifications thereof.
[0035] By "isolated nucleic acid" is meant a nucleic acid, e.g., a
DNA or RNA molecule, that is not immediately contiguous with the 5'
and 3' flanking sequences with which it normally is immediately
contiguous when present in the naturally occurring genome of the
organism from which it is derived. The term thus describes, for
example, a nucleic acid that is incorporated into a vector, such as
a plasmid or viral vector; a nucleic acid that is incorporated into
the genome of a heterologous cell (or the genome of a homologous
cell, but at a site different from that at which it naturally
occurs); and a nucleic acid that exists as a separate molecule,
e.g., a DNA fragment produced by PCR amplification or restriction
enzyme digestion, or an RNA molecule produced by in vitro
transcription. The term also describes a recombinant nucleic acid
that forms part of a hybrid gene encoding additional polypeptide
sequences that can be used, for example, in the production of a
fusion protein.
[0036] The nucleic acid molecules of the invention can be used as
templates in standard methods for production of FHF gene products
(e.g., FHF RNAs and FHF polypeptides; see below). In addition, the
nucleic acid molecules that encode FHF polypeptides (and fragments
thereof) and related nucleic acids, such as (1) nucleic acids
containing sequences that are complementary to, or that hybridize
to, nucleic acids encoding FHF polypeptides, or fragments thereof
(e.g., fragments containing at least 12, 15, 20, or 25
nucleotides); and (2) nucleic acids containing sequences that
hybridize to sequences that are complementary to nucleic acids
encoding FHF polypeptides, or fragments thereof (e.g., fragments
containing at least 12, 15, 20, or 25 nucleotides); can be used in
methods focused on their hybridization properties. For example, as
is described in further detail below; such nucleic acid molecules
can be used in the following methods: PCR methods for synthesizing
FHF nucleic acids, methods for detecting the presence of an FHF
nucleic acid in a sample, screening methods for identifying nucleic
acids encoding new FHF family members, and therapeutic methods.
[0037] The invention also includes methods for identifying nucleic
acid molecules that encode members of the FHF polypeptide family in
addition to FHFs 1-4. In these methods, a sample, e.g., a nucleic
acid library, such as a cDNA library, that contains a nucleic acid
encoding an FHF polypeptide is screened with an FHF-specific probe,
e.g., an FHF-specific nucleic acid probe. FHF-specific nucleic acid
probes are nucleic acid molecules (e.g., molecules containing DNA
or RNA nucleotides, or combinations or modifications thereof) that
specifically hybridize to nucleic acids encoding FHF polypeptides,
or to complementary sequences thereof. Because FHFs are closely
related to FGFs (i.e., the first nine members of the FGF family
(FGFs 1-9), see above), the-term "FHF-specific probe," in the
context of this method of invention, refers to probes that bind to
nucleic acids encoding FHF polypeptides, or to complementary
sequences thereof, to a detectably greater extent than to nucleic
acids encoding FGFs, or to complementary sequences thereof. The
term "FHF-specific probe" thus includes probes that can bind to
nucleic acids encoding FHF polypeptides (or to complementary
sequences thereof), but not to nucleic acids encoding FGFs (or to
complementary sequences thereof), to an appreciable extent.
[0038] The invention facilitates production of FHF-specific nucleic
acid probes. Methods for obtaining such probes can be designed
based on the amino acid sequence alignments shown in FIGS. 1-3. In
FIG. 1, for example, amino acid sequences that are conserved in
FHFs ("FHF-conserved amino acids") are boxed. In FIG. 2, amino
acids that are conserved in FHFs, but not in FGFs (i.e., FGFs 1-9)
("FHF-specific amino acids"), are indicated by large, black dots.
The probes, which can contain at least 12, e.g.,at least 15, 25,
35, 50, 100, or 150 nucleotides, can be produced using any of
several standard methods (see, e.g., Ausubel, et al., supra). For
example, preferably, the probes are generated using PCR
amplification methods, such as those described below in Example 1.
In these methods, primers are designed that correspond to
FHF-conserved sequences (FIG. 1), which can include FHF-specific
amino acids, and the resulting PCR product is used as a probe to
screen a nucleic acid library, such as a cDNA library. A nucleotide
sequence encoding FHF4 was identified generally following this
process based upon the analysis of the sequences of FHF 1-3.
[0039] As is known in the art, PCR primers are typically designed
to contain at least 15 nucleotides, for example 15-30 nucleotides.
The design of FHF-specific primers containing 21 nucleotides, which
encode FHF peptides containing 7 amino acids, are described as
follows. Preferably, most or all of the nucleotides in such a probe
encode FHF-conserved amino acids, including FHF-specific amino
acids. For example, primers containing sequences encoding peptides
containing at least 40% FHF-conserved amino acids can be used. Such
a primer, containing 21 nucleotides, can include sequences encoding
at least 3/7, 4/7, 5/7, 6/7, or 7/7 FHF-conserved amino acids. As
can be determined by analysis of FIGS. 1-3, in the case of a 21
nucleotide primer, encoding 7 amino acids, up to 5 amino acids can
be FHF-specific. Thus, the primer can contain sequences encoding at
least one FHF-specific amino acid, for example, up to 5
FHF-specific amino acids. Once FHF-specific amino acid sequences
are selected as templates against which primer sequences are to be
designed, the primers can be synthesized using, e.g., standard
chemical methods. As is described above, due to the degeneracy of
the genetic code, such primers should be designed to include
appropriate degenerate sequences, as can readily be determined by
one skilled in the art (see above, and Example 1, below).
[0040] Based on the guidelines presented above, examples of
FHF-conserved amino acid peptides that can be used as templates for
the design of FHF-specific primers are as follows. Additional
examples can be found by analysis of sequence alignments of FHF
polypeptides, for example, the alignments in FIGS. 1-3. Primers can
be designed, for example, based on 5-10 amino acid regions of these
peptides, depending on the lengths of the primers desired. For
example, primers can be designed to correspond to 7 consecutive
amino acids of any of the segments shown below.
1 1. A A A I/L A S S/G S L I R Q K R (SEQ ID NO:22) (corresponding
to amino acids 2-14 of human FHF-1) 2. P Q L K G I V T R/K (SEQ ID
NO:23) (corresponding to amino acids 68-76 of human FHF-1) 3. T L/H
F N L I P V G L R V V (SEQ ID NO:24) (corresponding to amino acids
104-116 of human FHF-1) 4. A M N G/S/A E G Y/L L Y (SEQ ID NO:25)
(corresponding to amino acids 128-136 of human FHF-1) 5. K E S/C V
F E N Y Y V (SEQ ID NO:26) (corresponding to amino acids 148-157 of
human FHF-1; see Example 1, below, for an example of a primer based
on the "V F E N Y Y V" (SEQ ID NO:27) portion of this sequence.) 6.
S G R A/G W F/Y L G L (SEQ ID NO:28) (corresponding to amino acids
169-177 of human FHF-1) 7. M K G N R/H V K K T I N K (SEQ ID NO:29)
(corresponding to amino acids 184-193 of human FHF-1; see Example
1, below, for an example of a primer based on the "M K G N H/R V K"
(SEQ ID NO:30) portion of this sequence.) 8. V C/A M Y R/Q/K E P S
L H (SEQ ID NO:31) (corresponding to amino acids 205-214 of human
FHF-1)
[0041] As is described above, FHF-specific primers, for example
primers based on the FHF-specific peptides shown above, or portions
thereof, can be used in PCR reactions to generate FHF-specific
probes, which can be used in standard screening methods to identify
nucleic acids encoding FHF family members (see, e.g., Ausubel, et
al., supra).
[0042] In addition to FHF-specific nucleic acid probes,
FHF-specific polypeptide probes, such as FHF-specific antibodies,
can be used to screen samples, e.g., expression libraries, for
nucleic acids encoding novel FHF polypeptides, or portions thereof.
For example, an antibody that specifically binds to an FHF-specific
peptide can be used in this method. Methods for carrying out such
screening are well known in the art (see, e.g., Ausubel, et al.,
supra).
[0043] The sequences of a pair of nucleic acid molecules (or two
regions within a single nucleic acid molecule) are said to be
"complementary" to each other if base pairing interactions can
occur between each nucleotide of one of the members of the pair and
each nucleotide of the other member of the pair. A pair of nucleic
acid molecules (or two regions within a single nucleic acid
molecule) are said to "hybridize" to each other if they form a
duplex by base pairing interactions between them. As is known in
the art, hybridization between nucleic acid pairs does not require
complete complementarity between the hybridizing regions, but only
that there is a sufficient level of base pairing to maintain the
duplex under the hybridization conditions used.
[0044] Hybridization reactions are typically carried out under low
to moderate stringency conditions, in which specific and some
non-specific interactions can occur. After hybridization, washing
can be carried out under moderate or high stringency conditions to
eliminate non-specific binding. As is known in the art, optimal
washing conditions can be determined empirically, e.g., by
gradually increasing the stringency. Condition parameters that can
be changed to affect stringency include, e.g., temperature and salt
concentration. In general, the lower the salt concentration and the
higher the temperature, the higher the stringency. For example,
washing can be initiated at a low temperature (e.g., room
temperature) using a solution containing an equivalent or lower
salt concentration as the hybridization solution. Subsequent
washing can be carried out using progressively warmer solutions
having the same salt solution. Alternatively, the salt
concentration can be lowered and the temperature maintained in the
washing step, or the salt concentration can be lowered and the
temperature increased. Additional parameters can be altered to
affect stringency, including, e.g., the use of a destabilizing
agent, such as formamide.
[0045] In nucleic acid hybridization reactions, the conditions used
to achieve a particular level of stringency will vary, depending on
the nature of the nucleic acids being hybridized. For example, the
length, degree of complementarity, nucleotide sequence composition
(e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA)
of the hybridizing regions of the nucleic acids can be considered
in selecting hybridization conditions. An additional consideration
is whether one of the nucleic acids is immobilized, for example, on
a filter. An example of progressively higher stringency conditions
is as follows: 2.times.SSC/0.1% SDS at about room temperature
(hybridization conditions); 0.2.times.SSC/0.1% SDS at about room
temperature (low stringency conditions); 0.2.times.SSC/0.1% SDS at
about 42.degree. C. (moderate stringency conditions); and
0.1.times.SSC at about 68.degree. C. (high stringency conditions).
Washing can be carried out using only one of these conditions,
e.g., high stringency conditions, or each of the conditions can be
used, e.g., for 10-15 minutes each, in the order listed above,
repeating any or all of the steps listed. However, as mentioned
above, optimal conditions will vary, depending on the particular
hybridization reaction involved, and can be determined
empirically.
[0046] The nucleic acid molecules of the invention can be obtained
by any of several standard methods. For example, the molecules can
be produced using standard recombinant, enzymatic (e.g., PCR or
reverse transcription (RT)/PCR methods), and chemical (e.g.,
phosphoramidite-based synthesis) methods. In addition, they can be
isolated from samples, such as nucleic acid libraries and tissue
samples, using standard hybridization methods. For example, as
described above, using standard methods, genomic or cDNA libraries
can be hybridized with nucleic acid probes corresponding to FHF
nucleic acid sequences to detect the presence of a homologous
nucleotide sequence in the library (see, e.g., Ausubel, et al.,
supra). These methods are described in more detail above. Also as
described above, nucleic acids encoding polypeptides containing at
least one FHF epitope, such as an FHF-specific epitope, can also be
identified by screening a cDNA expression library, such as a
library contained in lambda gt11, with an FHF-specific antibody as
a probe. Such antibodies can be either polyclonal or monoclonal and
are produced using standard methods (see, e.g., Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, New York, 1988).
[0047] The FHF nucleic acid molecules can be inserted into vectors,
such as plasmid or viral vectors, that facilitate (1) expression of
the inserted nucleic acid molecule and/or (2) amplification of the
insert. As is well known in the art, such vectors can contain,
e.g., promoter sequences, which facilitate transcription of the
inserted nucleic acid in the cell, origins of replication, and
genes, such as a neomycin-resistance gene, which encodes a
selectable marker that imparts G418 resistance to cells in which it
is expressed, and thus permits phenotypic selection of transformed
cells.
[0048] Vectors suitable for use in the present invention include,
e.g., T7-based expression vectors for use in bacteria (see, e.g.,
Rosenberg, et al., Gene, 56:125, 1987), the pMSXND expression
vector for use in mammalian cells (Lee and Nathans, J. Biol. Chem.,
263:3521, 1988), and baculovirus-derived vectors for use in insect
cells. The nucleic acids in such vectors are operably linked to a
promoter, which is selected based on, e.g., the cell type in which
expression is sought. For example, a T7 promoter can be used in
bacteria, a polyhedrin promoter can be used in insect cells, and a
cytomegalovirus or metallothionein promoter can be used in
mammalian cells. Also, in the case of higher eukaryotes,
tissue-specific promoters are available. (See, e.g., Ausubel, et
al., supra, for additional appropriate vectors and promoters that
can be used in the invention; also see Pouwels, et al., Cloning
Vectors: A Laboratory Manual, 1985, Supp. 1987). Viral vectors that
can be used -in the invention include, for example, retroviral,
adenoviral, adeno-associated viral, herpes virus, simian virus 40
(SV40), and bovine papilloma virus vectors (see, e.g., Gluzman ed.,
Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1982), and are discussed further below.
[0049] Cells into which FHF nucleic acids can be introduced, in
order to, for example, produce FHF polypeptides using, e.g., the
vectors described above, include prokaryotic cells (e.g., bacterial
cells, such as E. coli cells) and eukaryotic cells (e.g., yeast
cells, such as Saccharomyces cerevisiae cells; insect cells, such
as Spodoptera frugiperda cells (e.g., Sf-9 cells); and mammalian
cells, such as CHO, Cos-1, NIH-3T3, and JEG3 cells). Such cells are
available from a number of different sources that are known to
those skilled in the art, e.g., the American Type Culture
Collection (ATCC), Rockville, Md. (also see Ausubel, et al.,
supra). Cells into which the nucleic acids of the invention have
been introduced, as well as their progeny, even if not identical to
the parental cells, due to mutations, are included in the
invention.
[0050] Methods for introducing the nucleic acids of the invention
(e.g., nucleic acids inserted into the vectors described above)
into cells, either transiently or stably, are well known in the art
(see, e.g., Ausubel, et al., supra). For example, in the case of
prokaryotic cells, such as E. coli cells, competent cells, which
are prepared from exponentially growing bacteria using a standard
CaCl.sub.2 (or MgCl.sub.2 or RbCl) method, can be transformed using
standard methods. Transformation of bacterial cells can also be
performed using protoplast fusion methods. In the case of
eukaryotic cells, transfection can be carried out using calcium
phosphate precipitation or conventional mechanical procedures, such
as microinjection and electroporation, can be used. Also, the
nucleic acid (e.g., contained in a plasmid) can be packaged in a
liposome using standard methods. In the case of viral vectors,
appropriate infection methods, which are well known in the art, can
be used (see, e.g., Ausubel, et al., supra). In addition to being
transfected with a nucleic acid encoding an FHF polypeptide of the
invention, eukaryotic cells, such as mammalian cells, can be
co-transfected with a second nucleic acid encoding a selectable
marker, such as a neomycin resistance gene or the herpes simplex
virus thymidine kinase gene. As is mentioned above, such selectable
markers can facilitate selection of transformed cells.
[0051] Isolation and purification of polypeptides produced in the
systems described above can be carried out using conventional
methods, appropriate for the particular system. For example,
preparative chromatography and immunological separations employing
antibodies, such as monoclonal or polyclonal antibodies, can be
used.
[0052] Antibodies, such as monoclonal and polyclonal antibodies,
that specifically bind to FHF polypeptides (e.g., any or all of
FHFs 1-4) are also included in the invention. These antibodies can
be made by using an FHF polypeptide, or an FHF polypeptide fragment
that maintain an FHF epitope, as an immunogen in standard antibody
production methods (see, e.g., Kohler, et al., Nature, 256:495,
1975; Ausubel, et al., supra; Harlow and Lane, supra).
[0053] The term "antibody," as used herein, refers to intact
immunoglobulin molecules, as well as fragments of immunoglobulin
molecules, such as Fab, Fab', (Fab').sub.2, Fv, and SCA fragments,
that are capable of binding to an epitope of an FHF polypeptide.
These antibody fragments, which retain some ability to selectively
bind to the antigen (e.g., an FHF antigen) of the antibody from
which they are derived, can be made using well known methods in the
art (see, e.g., Harlow and Lane, supra), and are described further,
as follows.
[0054] (1) A Fab fragment consists of a monovalent antigen-binding
fragment of an antibody molecule, and can be produced by digestion
of a whole antibody molecule with the enzyme papain, to yield a
fragment consisting of an intact light chain and a portion of a
heavy chain.
[0055] (2) A Fab' fragment of an antibody molecule can be obtained
by treating a whole antibody molecule with pepsin, followed by
reduction, to yield a molecule consisting of an intact light chain
and a portion of a heavy chain. Two Fab' fragments are obtained per
antibody molecule treated in this manner.
[0056] (3) A (Fab').sub.2 fragment of an antibody can be obtained
by treating a whole antibody molecule with the enzyme pepsin,
without subsequent reduction. A (Fab').sub.2 fragment is a dimer of
two Fab' fragments, held together by two disulfide bonds.
[0057] (4) An Fv fragment is defined as a genetically engineered
fragment containing the variable region of a light chain and the
variable region of a heavy chain expressed as two chains.
[0058] (5) A single chain antibody ("SCA") is a genetically
engineered single chain molecule containing the variable region of
a light chain and the variable region of a heavy chain, linked by a
suitable, flexible polypeptide linker.
[0059] As used in this invention, the term "epitope" refers to an
antigenic determinant on an antigen, such as an FHF polypeptide, to
which the paratope of an antibody, such as an FHF4-specific
antibody, binds. Antigenic determinants usually consist of
chemically active surface groupings of molecules, such as amino
acids or sugar side chains, and can have specific three-dimensional
structural characteristics, as well as specific charge
characteristics.
[0060] As is mentioned above, antigens that can be used in
producing FHF-specific antibodies include FHF polypeptides, e.g.,
any of FHFs 1-4, or FHF polypeptide fragments. The polypeptide or
peptide used to immunize an animal can be obtained by standard
recombinant, chemical synthetic, or purification methods. As is
well known in the art, in order to increase immunogenicity, an
antigen can be conjugated to a carrier protein. Commonly used
carriers include keyhole limpet hemocyanin (KLH), thyroglobulin,
bovine serum albumin (BSA), and tetanus toxoid. The coupled peptide
is then used to immunize the animal (e.g., a mouse, a rat, or a
rabbit). In addition to such carriers, well known adjuvants can be
administered with the antigen to facilitate induction of a strong
immune response.
[0061] FHF-specific polyclonal and monoclonal antibodies can be
purified, for example, by binding to, and elution from, a matrix
containing an FHF polypeptide, e.g., the FHF polypeptide (or
fragment thereof) to which the antibodies were raised. Additional
methods for antibody purification and concentration are well known
in the art and can be practiced with the FHF-specific antibodies of
the invention (see, for example, Coligan, et al., Unit 9, Current
Protocols in Immunology, Wiley Interscience, 1994).
[0062] Anti-idiotype antibodies corresponding to FHF-specific
antigens are also included in the invention, and can be produced
using standard methods. These antibodies are raised to FHF-specific
antibodies, and thus mimic FHF-specific epitopes.
[0063] The members of a pair of molecules (e.g., an
antibody-antigen pair or a nucleic acid pair) are said to
"specifically bind" to each other if they bind to each other with
greater affinity than to other, non-specific molecules. For
example, an antibody raised against an antigen to which it binds
more efficiently than to a non-specific protein can be described as
specifically binding to the antigen. (Similarly, a nucleic acid
probe can be described as specifically binding to a nucleic acid
target if it forms a specific duplex with the target by base
pairing interactions (see above).)
[0064] As is discussed above, because of their amino acid sequence
homologies to previously identified FGF polypeptides (i.e., FGFs
1-9), as well as their tissue localizations, FHFs are thought to
play roles in regulating the development and function of the
nervous system. Altered levels of FHFs, such as increased levels,
may thus be associated with cell proliferative disorders, such as
cell proliferative disorders of the nervous system. The term
"cell-proliferative disorder" is used herein to describe conditions
that are characterized by abnormally excessive cell growth,
including malignant, as well as non-malignant, cell growth.
Conversely, conditions characterized by inadequate cell growth may
be characterized by decreased expression of FHFs. Accordingly,
these conditions can be diagnosed and monitored by detecting the
levels of FHFs in patient samples.
[0065] FHF-specific antibodies and nucleic acids can be used as
probes in methods to detect the presence of an FHF polypeptide
(using an antibody) or nucleic acid (using a nucleic acid probe) in
a sample, such as a biological fluid (e.g., cerebrospinal fluid
(CSF), such as lumbar or ventricular CSF) or a tissue sample (e.g.,
CNS tissue, e.g., neural tissue or eye tissue). In these methods,
an FHF-specific antibody or nucleic acid probe is contacted with a
sample from a patient suspected of having an FHF-associated
disorder, and specific binding of the antibody or nucleic acid
probe to the sample detected. The level of FHF polypeptide or
nucleic acid present in the suspect sample can be compared with the
level in a control sample, e.g., an equivalent sample from an
unaffected individual, to determine whether the patient has an
FHF-associated cell proliferative disorder. FHF polypeptides, or
fragments thereof, can also be used as probes in diagnostic
methods, for example, to detect the presence of FHF-specific
antibodies in samples.
[0066] The FHF-specific nucleic acid probes can be labeled with a
compound that facilitates detection of binding to the FHF nucleic
acid in the sample. For example, the probe can contain biotinylated
nucleotides, to which detectably labeled avidin conjugates (e.g.,
horse-radish peroxidase-conjugated avidin) can bind. Radiolabeled
nucleic acid probes can also be used. These probes can be used in
nucleic acid hybridization assays to detect altered levels of FHFs
in a sample. For example, in situ hybridization, RNAse protection,
and Northern Blot methods can be used. Other standard nucleic acid
detection methods that can be used in the invention are known to
those of skill in the art (see, e.g., Ausubel, et al., supra). In
addition, when the diagnostic molecule is a nucleic acid, it can be
amplified prior to binding with an FHF-specific probe. Preferably,
PCR is used, but other nucleic acid amplification methods, such as
the ligase chain reaction (LCR), ligated activated transcription
(LAT), and nucleic acid sequence-based amplification (NASBA)
methods can be used.
[0067] Use of FHF-specific antibodies in diagnostic methods is
described further, as follows. The antibodies of the invention can
be used in vitro or in vivo for immunodiagnosis The antibodies are
suited for use in, for example, immunoassays in which they are in
liquid phase or bound to a solid phase carrier (e.g., a glass,
polystyrene, polypropylene, polyethylene, dextran, nylon, amylase,
natural and modified cellulose, polyacrylamide, agarose, or
magnetite carrier). The antibodies used in such immunoassays can be
detectably labeled (e.g., with an enzyme, a radioisotope, a
fluorescent compound, a colloidal metal, a chemiluminescent
compound, a phosphorescent compound, or a bioluminescent compound)
using any of several standard methods that are well known in the
art. Examples of immunoassays in which the antibodies of the
invention can be used include, e.g., competitive and
non-competitive immunoassays, which are carried out using either
direct or indirect formats. Examples of such immunoassays include
radioimmunoassays (RIA) and sandwich assays (e.g., enzyme-linked
immunosorbent assays (ELISAs)). Detection of antigens using the
antibodies of the invention can be done using immunoassays that are
run in either forward, reverse, or simultaneous modes, including
immunohistochemical assays on physiological samples. Other
immunoassay formats are well known in the art, and can be used in
the invention (see, e.g., Coligan, et al., supra).
[0068] In addition to the in vitro methods described above,
FHF-specific monoclonal antibodies can be used in methods for in
vivo detection of an antigen, such as an FHF antigen (e.g., any one
of FHFs 1-4). In these methods, a detectably labeled antibody is
administered to a patient in a dose that is determined to be
diagnostically effective by one skilled in the art. The term
"diagnostically effective" is used herein to describe the amount of
detectably labeled monoclonal antibody that is administered in a
sufficient quantity to enable detection of the site having the
antigen for which the monoclonal antibody is specific. As would be
apparent to one skilled in the art, the concentration of detectably
labeled monoclonal antibody that is administered should be
sufficient so that the binding of the antibody to the cells
containing the polypeptide is detectable, compared to background.
Further, it is desirable that the detectably labeled monoclonal
antibody is rapidly cleared from the circulatory system, to give
the optimal target-to-background signal ratio.
[0069] The dosage of detectably labeled monoclonal antibodies for
in vivo diagnosis will vary, depending on such factors as the age
and weight of the individual, as well as the extent of the disease.
The dosages can also vary depending on factors such as whether
multiple administrations are intended, antigenic burden, and other
factors known to those of skill in the art.
[0070] In addition to initial diagnosis, the FHF polypeptides,
nucleic acids, and FHF-specific antibodies described above can be
used in in vitro or in vivo methods for monitoring the progress of
a condition associated with FHF expression. For example, they can
be used in methods to monitor the course of amelioration of an
FHF-associated disease, for example, after treatment has begun. In
these methods, changes in the levels of an FHF-specific marker
(e.g., an FHF polypeptide, an FHF nucleic acid, or an FHF-specific
antibody) are detected, either in a sample from a patient or using
the in vivo methods described above.
[0071] The invention also provides methods for treating conditions
associated with altered expression of FHF polypeptides, for
example, cell proliferative disorders (e.g., cell proliferative
disorders of the central or peripheral nervous systems, for
example, conditions affecting neural tissue, testes, heart tissue,
and cells of the eye). Treatment of an FHF-associated cell
proliferative disorder can be carried out, for example, by
modulating FHF gene expression or FHF activity in a cell. The term
"modulate" includes, for example, suppressing expression of an FHF
when it is over-expressed, and augmenting expression of an FHF when
it is under-expressed. In cases where a cell-proliferative disorder
is associated with over-expression of an FHF, nucleic acids that
interfere with FHF expression, at transcriptional or translational
levels, can be used to treat the disorder. This approach employs,
for example, antisense nucleic acids (i.e., nucleic acids that are
complementary to, or capable of hybridizing with, a target nucleic
acid, e.g., a nucleic acid encoding an FHF polypeptide), ribozymes,
or triplex agents. The antisense and triplex approaches function by
masking the nucleic acid, while the ribozyme strategy functions by
cleaving the nucleic acid. In addition, antibodies that bind to FHF
polypeptides can be used in methods to block the activity of an
FHF.
[0072] The use of antisense methods to inhibit the in vitro
translation of genes is well known in the art (see, e.g.,
Marcus-Sakura, Anal. Biochem., 172:289, 1988). Antisense nucleic
acids are nucleic acid molecules (e.g., molecules containing DNA
nucleotides, RNA nucleotides, or modifications (e.g., modification
that increase the stability of the molecule, such as 2'-O-alkyl
(e.g., methyl) substituted nucleotides) or combinations thereof)
that are complementary to, or that hybridize to, at least a portion
of a specific nucleic acid molecule, such as an RNA molecule (e.g.,
an mRNA molecule) (see, e.g., Weintraub, Scientific American,
262:40 1990). The antisense nucleic acids hybridize to
corresponding nucleic acids, such as mRNAs, to form a
double-stranded molecule, which interferes with translation of the
mRNA, as the cell will not translate an double-stranded mRNA.
Antisense nucleic acids used in the invention are typically at
least 10-12 nucleotides in length, for example, at least 15, 20,
25, 50, 75, or 100 nucleotides in length. The antisense nucleic
acid can also be as long as the target nucleic acid with which it
is intended that it form an inhibitory duplex. As is described
further below, the antisense nucleic acids can be introduced into
cells as antisense oligonucleotides, or can be produced in a cell
in which a nucleic acid encoding the antisense nucleic acid has
been introduced by, for example, using gene therapy methods.
[0073] Introduction of FHF antisense nucleic acids into cells
affected by a proliferative disorder, for the purpose of gene
therapy, can be achieved using a recombinant expression vector,
such as a chimeric virus or a colloidal dispersion system, such as
a targeted liposome. Those of skill in this art know or can easily
ascertain the appropriate route and means for introduction of sense
or antisense FHF nucleic acids, without resort to undue
experimentation.
[0074] Gene therapy methods can also be used to deliver genes
encoding FHF polypeptides (e.g., any of FHFs 1-4) to cells. These
methods can be carried out to treat conditions associated with
insufficient FHF expression. Thus, these methods can be used to
promote tissue repair or replacement, for example, in conditions
including stroke, neurodegenerative. diseases, such as Parkinson's
disease and Alzheimer's disease, retinal degenerative diseases,
such as retinitis pigmentosa and macular degeneration, and
peripheral neuropathies.
[0075] Due to the high levels of expression of FHF4 in the testes,
there are a variety of applications for FHF-4-specific
polypeptides, nucleic acids, and antibodies related to treating
disorders of this tissue. Such applications include treatment of
cell proliferative disorders related to FHF-4 expression in the
testes. Various testicular developmental or acquired disorders can
also be treated using FHF4-related molecules. These conditions
include, for example, viral infection (e.g., viral orchitis),
autoimmunity, sperm production or dysfunction, trauma, and
testicular tumors. The presence of high levels of FHF-4 in the
testes also suggests that FHF4, or an FHF4 analogue, can be used to
affect male fertility.
[0076] In addition to blocking mRNA translation, oligonucleotides,
such as antisense oligonucleotides, can be used in methods to stall
transcription, such as the triplex method. In this method, an
oligonucleotide winds around double-helical DNA in a
sequence-specific manner, forming a-three-stranded helix, which
blocks transcription from the targeted gene. These triplex
compounds can be designed to recognize a unique site on a chosen
gene (Maher, et al, Antisense Res. and Dev., 1(3):227, 1991;
Helene, Anticancer Drug Design, 6(6):569, 1991). Specifically
targeted ribozymes can also be used in therapeutic methods directed
at decreasing FHF expression.
[0077] The following examples are intended to illustrate, but not
to limit, the invention. While the procedures described in the
examples are typical of those that can be used to carry out certain
aspects of the invention, other procedures known to those skilled
in the art can also be used. The following materials and methods
were used in carrying out the experiments described in the
examples.
Materials and Methods
[0078] Random cDNA Sequencing. Details of retina cDNA library
construction, template preparation; and sequence determination are
described by Wang, et al., J. Biol. Chem. 271, 4468-4476, 1996.
[0079] Degenerate PCR. A fully degenerate sense strand primer, with
a flanking EcoRI restriction site, was synthesized to correspond to
the amino acid sequence VFENYYV (SEQ ID NO:27), and three partially
degenerate antisense primers, with a flanking BamHI site, were
synthesized to include all possible codons for the amino acid
sequence MKGN(H/R)VK (SEQ ID NO:30). These primers were used to
amplify human, murine, and bovine genomic DNA templates using T.
aquaticus polymerase under the following conditions:
1.times.(94.degree. C., 7 minutes), 35.times.(45.degree. C., 2
minutes; 72.degree. C., 0.5 minutes; 94.degree. C., 0.5 minutes;
95.degree. C., 0.25 minutes). PCR products were cleaved with EcoRI
and BamHI, fractionated by preparative agarose gel electrophoresis,
subcloned into pBluescript, and sequenced individually.
[0080] cDNA and Genomic Clones. Oligo-dT primed cDNA libraries from
adult human retinas (Nathans, et al., Science 232, 193-202, 1986)
and P0-P7 mouse eyes were screened by DNA hybridization under
standard conditions (see, e.g., Sambrook, et al., Molecular
Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989). The complete
coding region sequences of human FHF-1, FHF-2, and FHF-3, and mouse
FHF-1, FHF-3, and FHF-4 were obtained from two independent cDNA
clones. For human FHF-4, the first 72 codons were sequenced from a
single clone, and the rest of the coding-region was sequenced from
two independent clones. For mouse FHF-2, the coding sequence was
determined from cloned genomic DNA and from a PCR product obtained
by amplification of the full-length, coding region from the P0-P7
mouse eye cDNA library. A partial MboI digest of a mouse genomic
DNA library in bacteriophage lambda was screened to obtain the
mouse FHF-2 genomic clones.
[0081] Chromosomal Localization. Human chromosome mapping was
performed by Southern blot analysis of DNA obtained from a panel of
24 human-mouse or human-hamster hybrid cell lines each carrying a
different human chromosome (Oncor, Gaithersburg, Md.).
Interspecific backcross progeny were generated by mating
(C57BL/6J.times.M. spretus) F1 females and C57BL/6J males, as
described (Copeland and Jenkins, Trends Genet. 7,113-118, 1991). A
total of 205 N2 mice were used to map the FHF loci. DNA isolation,
restriction enzyme digestion, agarose gel electrophoresis, Southern
blot transfer, and hybridization were performed essentially as
described (Jenkins, et al., J. Virol 43, 26-36, 1982), using
Zetabind nylon membranes (AMF-Cuno). Washing was carried out to a
final stringency of 0.8-1.0.times.SSCP, 0.1% SDS, at 65.degree. C.
The FHF-1 probe, a 1 kilobase fragment of mouse genomic DNA,
detected fragments of 2.1 kilobases in C57BL/6J (B) DNA and 10.0
kilobases in M spretus (S) DNA, following digestion with BamHI. The
FHF-2 probe, a 0.75 kilobase fragment of mouse cDNA, detected BglI
fragments of 19.0, 11.5, 8.2, and 2.2 kilobases (B) and 13.5, 8.2,
and 2.2 kilobases (S). The FHF-4 probe, a 0.3 kilobase fragment of
mouse cDNA, detected EcoRV fragments of approximately 24.0
kilobases (B) and 7.7 kilobases (S).
[0082] Most of the probes and RFLPs for the loci linked to the FHF
genes in the interspecific backcross have been reported earlier.
These include: Irg1 and Rap2a on chromosome 14 (Lee, et al.,
Immunogenet. 41, 263-270, 1995); Smst on chromosome 16 (Siracusa,
et al., Genetics 127, 169-179, 1991); and Hprt, Cd401, and Ar on
the X chromosome (Allen, et al., Science 259, 990-993, 1993;
Fletcher, et al., Genomics 24, 127-132, 1994). The probe for
apolipoprotein D (Apod), a 290 base pair HindIII/BamHI fragment of
a rat cDNA that was provided by Alan Peterson, detected XbaI
fragments of 3.1 and 2.8 kilobases (B) and 3.1 and 2.6 kilobases
(S). The inheritance of the 2.6 kilobase M. spretus-specific XbaI
RFLP was followed. Recombination distances were calculated as
described (Green, in Genetics and Probability in Animal Breeding
Experiments (Oxford Press, New York), pp. 77-113, 1981) using the
computer program SPRETUS MADNESS. Gene order was determined by
minimizing the number of recombination events required to explain
the allele distribution patterns.
[0083] RNAse Protection. Total RNA was prepared from adult mouse
brain, eye, heart, kidney, liver, lung, spleen, and testis by
homogenization in guanidinium thiocyanate and extraction with
phenol, followed by centrifugation through 5.7 M cesium chloride
(Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989). Ten micrograms of total RNA from each tissue, or ten
micrograms of yeast tRNA, was used for the RNAse protection assay.
Riboprobes were synthesized using either T7 or T3 RNA polymerase on
linearized templates that were cloned in pBluescript. Each mouse
FHF probe contained 150-250 nucleotides from the antisense strand,
linked to 25-50 nucleotides of vector sequence. Reagents were
obtained from Ambion (Austin, Tex.), and the hybridization and
digestion conditions used were as recommended by Ambion.
[0084] In Situ Hybridization. Freshly dissected adult mouse brains,
whole embryos, or heads were rapidly frozen in plastic molds placed
on a dry ice/ethanol slurry and processed for sectioning as
previously described (Cole, et al., J. Neurochem. 55, 1920-1927,
1990). .sup.33P-labeled antisense riboprobes were prepared from
linearized pBluescript plasmid subclones, using either T3 or T7 RNA
polymerase. In situ hybridization was performed in 50% formamide,
0.3 M NaCl at 56.degree. C., as described (Saffen, Proc. Natl.
Acad. Sci. USA 85, 7795-7799, 1988). Following RNAse treatment, the
slides were washed for 1 hour in 0.1.times.SSC at 55.degree. C.
After the hybridized sections were exposed to X-ray film, the
slides were stained with cresyl violet. Digitized images of the
stained slides and corresponding autoradiograms were superimposed
using Adobe Photoshop software. The probes used were: FHF-1, 0.75
kilobase containing the complete coding region; FHF-2, 0.5 kilobase
containing 0.3 kilobase of intron 4 and 0.2 kilobase of exon 5;
FHF-3, two 0.4 kilobase segments containing the 5' or 3' halves of
the coding region; and FHF4, 0.5 kilobase containing the 3'
two-thirds of the coding region. The coding regions of the
different murine FHFs share between 63% and 71% nucleotide sequence
identity, suggesting that there should be little or no
cross-hybridization under the conditions used.
[0085] Immunohistochemistry. Rabbit polyclonal anti-FHF-1
antibodies were raised against a bacterial fusion protein
consisting of the carboxyl-terminal 190 amino acids of FHF-1 fused
to the T7 gene 10 protein (Studier, et al., Meth. Enzymol. 185,
60-89, 1980). Anti-FHF-1 antibodies were affinity purified using
the fusion protein immobilized on nitrocellulose as an affinity
matrix, and antibodies directed against the fusion partner were
removed by absorption onto immobilized T7 gene 10 protein. For
immunostaining of primate brain samples, three monkeys (Macaca
mulatta) were anesthetized with Ketamine (35 mg/kg, i.m.), injected
with a lethal dose of sodium pentobarbital (100 mg/kg, i.v.), and
perfused through the heart with 4% paraformaldehyde in 0.1 M
phosphate buffer (pH 7.4). The brains were removed, cryoprotected
in 20% sucrose at 4.degree. C., frozen, and cut at a thickness of
10 or 20 .mu.m. Sections were sequentially incubated with affinity
purified-rabbit anti-FHF-1 antibodies, biotinylated goat
anti-rabbit IgG (Vector), peroxidase-conjugated avidin (Extravidin,
Sigma Chemical Co.), and 3,3'-diaminobenzidine dihydrochloride
(Aldrich), in the presence of hydrogen peroxide. Most sections were
then mounted on gelatin-coated slides, dehydrated, cleared, and
covered with a cover-slip. Following the histochemical reaction,
some of the 10 .mu.m-thick sections were washed and incubated
sequentially in mouse anti-parvalbumin (Sigma), biotinylated horse
anti-mouse IgG (Vector), and streptavidin conjugated to Texas Red
(Chemicon). These sections were mounted on clean slides, partially
dried, and covered in a glycerol/phosphate buffer medium. Adjacent
sections were processed histochemically for cytochrome oxidase or
were stained for Nissl substance, to determine the boundaries of
subcortical nuclei or cortical areas and layers.
[0086] Production and Localization of FHF-1 in Transfected Cells.
To increase the efficiency of FHF-1 translation, the region
immediately 5' to the initiator methionine coding sequence was
converted to an optimal ribosome binding site (CCACCATGG) by PCR
amplification, before inserting the complete FHF-1 open reading
frame into the eukaryotic expression vector pCIS (Gorman, et al.,
DNA Protein Eng. Tech. 2 3-10, 1990). The pCIS vector was also used
for the beta-galactosidase constructs. Human embryonic kidney cells
(293 cells) were transiently transfected with the expression
construct and a plasmid expressing the simian virus 40 (SV40) large
T-antigen (pRSV-TAg) using the calcium phosphate method (Gorman, et
al., supra). For .sup.35S-methionine labeling, cells were
transferred to serum-free medium 24 hours after transfection, and
labeled for 6 hours. For immunostaining, transfected cells were
grown on gelatin-coated coverslips. One day after transfection, the
cells were fixed in 2% formaldehyde in PBS for 30 minutes at room
temperature, permeabilized with cold methanol for 10 minutes at
-20.degree. C., preincubated for 15 minutes in 3% BSA in
phosphate-buffered saline (PBS), incubated for 1 hour at room
temperature with affinity purified anti-FHF-1 antibody at a
dilution of 1:1000 and mouse monoclonal anti-BiP at a dilution of
1:400 in 3% BSA in PBS, washed 3.times.15 minutes with 0.1%
TWBEN-20.TM. in PBS at room temperature, and then incubated with
fluorescein-conjugated donkey anti-rabbit IgG and
rhodamine-conjugated goat anti-mouse IgG in 3% BSA in PBS for 30-60
minutes. The coverslips were then washed in 0.1% TWEEN-20.TM. in
PBS and mounted in 0.1% 1,4-diazobicyclo-[2,2,2]-octane (DABCO),
75% glycerol, 10 mM Tris, pH 8.0. For X-gal staining, transfected
cells were fixed in 0.5% glutaraldehyde/PBS for 10 minutes at room
temperature, washed twice in PBS with 2 mM MgCl.sub.2, incubated in
1 mg/ml 5 mM K.sub.3Fe(CN).sub.6, 5 mM K.sub.4Fe(CN).sub.6, 2 mM
MgCl.sub.2 in PBS for 1-2 hours at 37.degree. C. and in 0.5%
glutaraldehyde in PBS for 10 minutes at room temperature.
EXAMPLE 1
Identification of Fibroblast Growth Factor Homologous Factors
(FHFs)
[0087] To identify gene products expressed in the human retina,
random segments of human retina cDNA clones were partially
sequenced, and the resulting partial sequences were compared to
sequences in publicly available databases. In detail, an adult
human retina cDNA library constructed in lambda gt10 (Nathans, et
al., Science, 232:193, 1986) was amplified, and the cDNA inserts
were excised en mass by cleavage with EcoRI and purified from the
vector by agarose gel electrophoresis. Following heat denaturation
of the purified cDNA inserts, a synthetic oligonucleotide having an
EcoRI site at its 5' end and six random nucleotides at its 3' end
(5'-GACGAGATATTAGAATTCTACTCGNNNNN-3'; (SEQ ID NO:32)) was used to
prime two sequential rounds of DNA synthesis in the presence of the
Kienow fragment of E. Coli DNA polymerase. The resulting duplex DNA
molecules were amplified by the polymerase chain reaction (PCR)
using a primer corresponding to the unique 5' flanking sequence
(5'-CCCCCCCCCGACGAGATATTAGAATTCTACTCG-3'; (SEQ ID NO:33)). The PCR
products, representing a random sampling of the original cDNA
inserts, were cleaved with EcoRI, size fractionated by preparative
agarose gel electrophoresis to include only segments of
approximately 500 base pairs in length, and cloned into lambda
gt10. Three thousand single plaques from this library were arrayed
in 96-well trays and the inserts from these clones were amplified
by PCR and then sequenced using the dideoxy method and automated
fluorescent detection (Applied Biosystems). A single sequencing run
from one end of each insert was conceptually translated for both
strands, in all three reading frames, and the six resulting amino
acid sequences were used to search for homology in the GenBank
nonredundant protein database using the BLASTX searching
algorithm.
[0088] One partial cDNA sequence was identified that showed
statistically significant homology to previously described members
of the FGF family. Using this partial cDNA as a probe, multiple,
independent cDNA clones were isolated from the human retina cDNA
library, including two clones that encompass the entire open
reading frame, and from which complete nucleotide sequences were
determined. The complete nucleotide sequence encodes a novel and
highly divergent member of the FGF superfamily, and was designated
fibroblast growth factor homologous factor-1 (FHF-1). The deduced
amino acid sequence of FHF-1 contains 244 amino acids and is 27%
identical to FGF-9, which is the member of the FGF family that
shares the most homology with FHF-1. The nucleotide and deduced
amino acid sequences of FHF-1 are shown in FIG. 5.
[0089] The FHF-1 sequence was used to search the National Center
for Biotechnology Information (NCBI) database of expressed sequence
tags (ESTs) and sequence tagged sites (STSs). One EST entry (DBEST
ID 06895), derived from a human infant brain cDNA library (Adams,
et al., Nature Genet. 4, 373-380, 1993) encoded a segment of 77
amino acids having significant homology to the carboxyl-terminus of
FHF-1, but no significant homology to other members of the FGF
family. The polypeptide containing this amino acid segment was
designated FHF-2. One STS entry (DBEST ID 76387; Brody, et al.,
Genomics 25, 238-247, 1995), derived from human genomic DNA
contained a putative exon encoding a protein segment with a high
degree of homology to FHF-1 and a low degree of homology to other
FGF family members. The polypeptide containing this amino acid
segment was designated FHF-3. Full-length FHF-2 and FHF-3 cDNA
clones were isolated from an adult human retina cDNA library, and
were found to encode proteins of 245 and 225 amino acids,
respectively, each having greater than 58% amino acid identity to
FHF-1 and to each other. The nucleotide and deduced amino acid
sequences of FHF-2 and FHF-3 are shown in FIG. 6 and FIG. 7,
respectively.
[0090] A comparison of the sequences of FHF-1, FHF-2, and FHF-3
revealed several regions of high amino acid sequence conservation,
and two of these regions were used to design degenerate
oligonucleotide primers for use in PCR. The primers have sequences
that correspond to codons for the conserved amino acids at their 3'
ends (at residues 151-157 and 184-190 in the FHF-1 sequence (SEQ ID
NO:1); see FIG. 5, and below), and restriction enzyme cleavage
sites at their 5' ends, to facilitate cloning of the resulting PCR
products. A full degenerate sense strand primer with a flanking
EcoRI restriction site was synthesized for the amino acid sequence
VFENYYV (SEQ ID NO:27; amino acids 151 -157)
(5'-CCGATCGAATTCGTNTT(T/C)GA(A/G)AA(T/C)TA(T/C)TA (T/C)GT-3'; SEQ
ID NO:34). Three partially degenerate antisense primers with a
flanking BamHI site were synthesized to include all possible codons
for the amino acid sequence MKGN(HIR)VK (SEQ ID NO:30; amino acids
184-190)
[0091] (5'-GCGATCGGATCCTTNAC(A/G)TG (A/G)TTNCC(T/C)TTCAT-3' (SEQ ID
NO:35);
[0092] 5'-GCGATCGGATCCTTNAC(T/C)CT(A/G)TTN CC(T/C)TTCAT-3' (SEQ ID
NO:36); and
[0093] 5'-GCGATCGGATCCTTNACNCG(A/G)TTNCC(T/C) TTCAT-3' (SEQ ID
NO:37)). The three pairs of sense and anti-sense primers were used
in PCR reactions containing human, murine, and bovine genomic DNA
templates and Thermus aquatics DNA polymerase and the reactions
were carried out under the following conditions:
1.times.(94.degree. C., 7 minutes), 35.times.(45.degree. C., 2
minutes; 72.degree. C., 0.5 minutes; 94.degree. C., 0.5 minutes;
95.degree. C., 0.25 minutes). PCR products were cleaved with EcoRI
and BamHI, fractionated by preparative agarose gel electrophoresis,
subcloned into pBluescript, and individually sequenced. Analysis of
the amplification products obtained using mouse, human, and bovine
genomic DNA templates revealed that, in each of these species, this
region of FHF-1, FHF-2, and FHF-3 is encoded within a single exon.
This analysis also revealed a fourth class of FHF-like PCR products
which was present in all three species and was found to encode an
FHF-like protein, designated FHF-4. The PCR product corresponding
to FHF-4 was used as a probe to isolate full-length cDNA clones
from a human retina cDNA library and a developing mouse eye cDNA
library. A protein of 247 amino acids having greater than 60% amino
acid identity to FHF-1, FHF-2, and FHF-3, is predicted to be
encoded by these clones. The nucleotide and deduced amino acid
sequence of FHF-4 is shown in FIG. 8.
[0094] FIG. 3 shows an alignment of the amino acid sequences of
identified FHFs and previously characterized and published FGFs
(1-9) and FIG. 4 is a dendrogram of the identified FHFs and all the
published FGF sequences. Pairwise comparisons between each FHF and
the nine FGF family members show less than 30% amino acid sequence
identity, while all pairwise comparisons among the four FHFs show
between 58% and 71% amino acid sequence identity. Between mouse and
human orthologues, there is greater than 97% amino acid sequence
identity. The murine FHFs differ from their human orthologues by
the amino acid substitutions listed below. The first and last
letters indicate the amino acids in the human and mouse sequences,
respectively, and the number indicates the position along the
polypeptide chain: FHF-1: Q86E; FHF-2: A2T, L136H; FHF-3: A58T,
P60Q, Q180R, L197V, Q207R, A217T, P225H; and FHF-4: C40F, A181V,
P221 A, S244C. Thus, the four FHFs define a distinct and highly
conserved branch of the FGF family.
[0095] FHFs 1-4 each lack a recognizable amino-terminal signal
sequence. Among the nine previously characterized members of the
FGF family, FGF-1, FGF-2, and FGF-9 are also distinguished by
lacking a recognizable amino-terminal signal sequence (Abraham, et
al., Science 233, 545-548, 1986; Jaye, et al., Science 233,
541-545, 1986; Miyamoto, et al., Mol. Cell Biol. 13, 4251-4259,
1993). Current evidence indicates that FGF-1 and FGF-2 are
synthesized in the cytosol and are released by a mechanism
independent of the ER-Golgi secretory pathway (Florkiewicz, et al.,
J. Cell Physiol. 162, 388-399, 1995; Jackson, et al., J. Biol.
Chem. 270, 33-36, 1995). Although FGF-9 lacks a cleavable
amino-terminal signal sequence, it is glycosylated and is
efficiently secreted from cultured glioma, Chinese hamster ovary,
and COS cells, presumably via the ER-Golgi pathway (Miyamoto, et
al., Mol. Cell Biol. 13, 4251-4259, 1993).
EXAMPLE 2
Deduced Amino Acid Sequence of FHF-4
[0096] FIG. 8 shows the nucleotide and deduced amino acid sequences
of human FHF4, which was derived from the human retina cDNA clones
described above. As is mentioned above, the primary translation
product of the human FHF-4 gene is predicted to be 247 amino acids
in length. The human FHF-4 initiator methionine codon shown in FIG.
8 at nucleotide positions 78-80 and is the first in frame ATG; a
good consensus ribosome binding site (CCACCATGG; Kozak, Nucleic
Acids Res., 15:8125, 1987) is found at this position. This choice
of ATG conforms to the sites of translation initiation in FHF-1,
FHF-2, and FHF-3, as shown in the alignment in FIG. 1. The next
methionine codon in the FHF-4 open reading frame is located 85
codons 3' to the putative initiator methionine codon. Similar to
FGF-1 and FGF-2, as well as FHFs 1-3, FHF-4 lacks a discernable
amino-terminal signal sequence. Human FHF-4 has a single
Asn-Lys-Ser motif at amino acids 242-244, which conforms to the
consensus sequence for asparagine-linked glycosylation, but this
site is not conserved in the highly homologous mouse FHF-4 sequence
(see FIG. 3), suggesting that it may not be used for
glycosylation.
EXAMPLE 3
Chromosomal Localization of FHF Genes
[0097] In humans, FHF-1, FHF-2, FHF-3, and FHF-4 are located on
chromosomes 3, X, 17, and 13, respectively. The chromosomal
locations of FHF-1, FHF-2, and FHF-4, were determined by Southern
blot hybridization of genomic DNA from rodent-human hybrid cell
lines carrying individual human chromosomes. For example, in the
case of human FHF-4, a Southern blot containing restriction
enzyme-digested DNA from a panel of 24 human-mouse and
human-hamster cell line, each containing a different human
chromosome (Oncor, Gaithersburg, Md.). Hybridization of a human
FHF-4 probe to human, mouse, and hamster genomic DNA produced
distinct hybridizing fragment sizes. Among the hybrid panels, the
human-specific hybridization pattern was seen in the lanes
corresponding to the hybrid cell line carrying human chromosomes 1
and 13, suggesting that one of these cell lines contains additional
genomic sequences derived from the chromosome present in the other
cell line. To determine which of these two human chromosomes
contained the FHF-4 gene, the location of the mouse gene was
determined by interspecific backcross mapping. As discussed further
below, this analysis located the FHF-4 gene on mouse chromosome 14,
less than 1 cM from the Rap2a gene, and within a region that is
syntenic with human chromosome 13q34. Taken together, these data
show that in humans, the FHF-4 gene maps to chromosome 13. The
FHF-3 locus is on human chromosome 17, as the STS described above
that encompasses one exon of FHF-3 was derived from human
chromosome 17 and maps near the BRCA-1 gene (Brody, et al.,
Genomics 25, 238-247, 1995).
[0098] The chromosomal locations of FHF-1, FHF-2, and FHF-4 in the
mouse were determined using an inter-specific backcross mapping
panel from crosses of (C57BL/6J.times.Mus spretus),
F1.times.C57BL/6J. This mapping panel has been typed for over 2100
loci, which are well distributed over all 19 mouse autosomes and
the X-chromosome (Copeland and Jenkins, Trends Genet. 7, 113-118,
1991). C57BL/6J and M. spretus DNAs were digested with several
restriction enzymes and analyzed by Southern blot hybridization for
informative RFLPs. The chromosomal location of each locus was
determined by comparing its strain distribution in the backcross
mice with the strain distribution patterns for all other loci
already mapped in the backcross (FIG. 9). FHF-1 mapped to the
proximal region of mouse chromosome 16, 1.6 cM distal to Smst and
5.1 cM proximal to Apod. FHF-2 mapped to the X chromosome, and did
not recombine with Cd41 in 168 mice typed in common, suggesting
that the two loci are within 1.8 cM of each other (upper 95%
confidence interval). As is mentioned above, FHF-4 mapped to the
distal region of chromosome 14, and did not recombine with Rap2a in
142. mice typed in common, suggesting that the two loci are within
2.1 cM of each other. The FHF-3 gene was not mapped with the
backcross panel, as it did not reveal an informative RFLP when
tested with 14 restriction enzymes. The proximity of the human
FHF-3 gene to BRCA-1 suggests that the mouse FHF-3 gene resides on
chromosome 11 in the region that is syntenic with the BRCA-1 region
of human chromosome 17. The FHF genes map in regions of the
composite mouse linkage map (Mouse Genome Database, Jackson
Laboratory, Bar Harbor, Me.), which contains a number of mutations
that may be candidate FHF alleles.
EXAMPLE 4
Tissue and Subcellular Distributions of FHF RNAs
[0099] Expression Patterns of FHFs in the Mouse. The tissue
distributions of transcripts derived from each of the four
identified FHF genes were determined in RNAse protection
experiments. Analysis of RNA prepared from brain, eye, heart,
kidney, liver, lung, spleen, and testis revealed that each FHF is
expressed in the brain, and FHF-1 FHF-2, and FHF-3 are expressed in
the eye, FHF-1 and FHF-4 are expressed in the testis, and FHF-2 is
expressed in the heart (FIG. 10). In the brain, FHF-2 transcripts
are at least five-fold more abundant than the transcripts from any
of the other FHFs.
[0100] The patterns of FHF gene expression during development were
studied by in situ hybridization experiments using sections
obtained on gestational day 11 (e11), gestational day 17 (e 17),
postnatal day 1 (P1), and from adults. FHF-2, transcripts are
abundant at each of the time points examined, and are present in
all divisions of the central and peripheral nervous systems,
including the enteric nervous system (FIGS. 11A, 11C, 11D, and
11F). Consistent with the RNAse protection experiments, FHF-2
transcripts were observed in the developing heart at e17 (FIG.
11D). FHF-1, FHF-3, and FHF-4 transcripts were also observed to be
widely distributed throughout the developing nervous system (FIGS.
11B and 11E). For example, at P1, FHF-1 was found to be highly
expressed in the retina, olfactory epithelium, and olfactory bulb
(FIG. 11E). At e11, FHF-1 and FHF-3 were also found in a segmental
pattern in the body wall (FIG. 11B). In the adult brain, each FHF
showed a distinct pattern of expression: FHF-1 transcripts were
present at high levels in the olfactory bulb, and at lower levels
in the cerebellum, the deep cerebellar nuclei, throughout the
cortex, and in multiple midbrain structures (FIG. 11G); FHF-2
transcripts were most abundant in the hippocampus and were present
at lower levels in multiple brain areas (FIGS. 11H and 11I); FHF-3
transcripts were present in the olfactory bulb, hippocampus, and
cerebellum, where they were most concentrated in the Purkinje cell
layer (FIG. 11J); and FHF-4 transcripts are present at high levels
throughout the granular layer of the cerebellum, and at lower
levels in the hippocampus and olfactory bulb (FIGS. 11K and
11L).
[0101] Distribution of FHF-1 Immunoreactivity in Monkey Brain. The
distribution of FHF-1 immunoreactivity in adult rhesus monkey brain
was examined using affinity purified polyclonal anti-FHF-1
antibodies. These antibodies bind to recombinant FHF-1, but do not
bind to recombinant FHF-2, as was determined by immunostaining of
transfected 293 cells and by Western blotting. Although
immunoreactivity with these antibodies is referred to as `FHF-1
immunoreactivity` the possibility exists that other members of the
FHF family are in part responsible for the observed
immunostaining.
[0102] FHF-1 immunoreactive somata are present throughout the
rhesus monkey cerebral cortex, but they are unevenly distributed
across layers in any one area and display marked variations in
density and distribution across functional areas (FIGS. 12A-12E).
The low magnification photomicrographs in FIG. 12 show that in
primary visual, somatosensory, and auditory areas, a relatively
high density of immunostained cells is present and that these cells
occupy predominantly the middle layers. In contrast, fewer and more
widely scattered immunostained neurons are present in the
precentral motor area and in the association cortex of the superior
parietal lobule.
[0103] Common to each of these cortical areas is the presence of
several FHF-1 immunoreactive populations, the most prominent of
which have relatively large (12-14 .mu.m diameter), intensely
immunoreactive somata. Other neurons with smaller (8-10 .mu.m
diameter) and more lightly immunostained somata are also present in
all areas (FIG. 12F). Co-localization experiments demonstrated that
the FHF-1 immunoreactive neurons in the cerebral cortex make up a
subpopulation of neurons that are immunoreactive for the
calcium-binding protein, parvalbumin (FIGS. 12G and 12H), which
have previously been shown to make up a subset of gabanergic
interneurons in the monkey cerebral cortex (Hendry, et al., Exp.
Brain Res. 76, 467-472, 1989). Variations in immunostained cell
density were also seen in the hippocampal formation, where
intensely immunoreactive somata were relatively common in the
subicular complex, but were widely scattered in the CA fields, the
dentate hilus, and the dentate gyrus (FIG. 12I).
[0104] A different pattern of FHF-1 immunostaining was seen in the
dorsal thalamus (FIG. 12J). Only a few of the many nuclei in this
region contained immunoreactive neurons; most prominent among them
was the principal somatosensory relay nucleus (the caudal
ventroposterolateral nucleus, VPLc) and the visual relay nucleus
(the lateral geniculate nucleus, LGN). In the LGN, both
magnocellular and parvicellular layers are equally immunostained.
FIG. 12J shows that in the LGN, ipsilateral to an eye deprived by
occlusion since birth, FHF-1 immunostaining was markedly lower in
layers innervated by the deprived eye than in layers innervated by
the normal eye.
[0105] The relatively large sizes of the FHF-1 immunostained somata
in nuclei of the dorsal thalamus suggests they are cell bodies of
neurons that send their axons to the cerebral cortex. Localization
of FHF-1 immunoreactivity in neurons of VPLc and LGN that are
lightly immunoreactive for parvalbumin supports this conclusion,
since these parvalbumin immunostained neurons in dorsal thalamus
have been shown to be thalamocortical neurons (Jones and Hendry,
Eur. J Neurosci. 1, 222-246, 1989).
[0106] Outside of the dorsal thalamus, FHF-1 immunoreactive neurons
were present in a diverse collection of subcortical nuclei,
including the globus pallidus and putamen, red nucleus, substantia
nigra, and third nerve complex (FIG. 12K). In addition, large cells
in the deep layers of both the superior and inferior colliculi were
immunostained, as were neurons in the deep cerebellar nuclei. In
conclusion, FHF-1 immunoreactivity was broadly distributed across
the neuraxis, but at each level it was present in subsets of
neurons.
[0107] Subcellular Localization of FHF-1 in Transfected Cells. As
noted above, of the nine FGFs described prior to this report, FGF-1
and FGF-2 are distinguished by lacking of an amino terminal signal
sequence and by secretion via a pathway that is independent of the
ER and Golgi apparatus (Florkiewicz, et al., J. Cell Physiol. 162,
388-399, 1995; Jackson, et al., J. Biol. Chem. 270, 33-36, 1995).
Moreover, FGF-1 and a subset of FGF-2 isoforms, produced by
alternative translation initiation, have been shown to accumulate
in the nuclei of the cells in which they are synthesized, as well
as in the nuclei of target cells (Imamaura, et al., Science 249,
1567-1570, 1990; Imamaura, et al., J. Biol. Chem. 267, 5676-5679,
1992; Bugler, et al., Molec. Cell Biol. 11, 573-577, 1991; Zhan, et
al., Biochem. Biophys. Res. Comm. 188, 982-991, 1992; Cao, et al.,
J. Cell Science 104, 77-87, 1993; Wiedlocha, et al., Cell 76,
1039-1051, 1994). These proteins contain a nuclear localization
signal (NLS) that conforms closely to a consensus NLS. The nuclear
accumulation of FGF-1 and FGF-2 has raised the possibility that
these proteins have modes of action in addition to those mediated
by binding and-activating cell surface receptors.
[0108] Like FGF-1 and FGF-2, each of the four identified FHFs lacks
a classical signal sequence and contains clusters of basic residues
near its amino terminus that could serve as an NLS. These
observations suggest that FHFs may resemble FGF-1 and FGF-2 in
their mechanism of secretion and in their subcellular localization.
To test this possibility, the fate of FHF-1 synthesized in
transiently transfected 293 cells, which are human embryonic kidney
cells, was determined. In one set of experiments, biosynthetically
labeled FHF-1 was found to accumulate to high levels
intracellularly, but was not detectably secreted into the medium
(FIG. 13). In contrast, human growth hormone produced in parallel
transfections was efficiently secreted (FIG. 13). As 293 cells can
secrete a wide variety of growth factors through the ER-Golgi
pathway, this experiment is consistent with the proposal that FHFs
are not secreted via this route.
[0109] In a second set of experiments, immunostaining of
transfected 293 cells revealed accumulation of FHF-1 in the
nucleus, suggesting that the clusters of basic residues can
function as an NLS. A similar result was obtained with FHF-2. Two
main classes of NLS motifs have been described, the classical and
bipartite motifs (Boulikas, Crit. Rev. Eukaryotic Gene Expression
3, 193-227, 1993). The classical NLS contains a cluster of six
lysine or arginine amino acids, and the bipartite NLS contains two
clusters of three or four basic amino acids separated by a ten
amino acid spacer. In FHF-1 the clusters of basic amino acids
resemble more closely the bipartite NLS consensus. To identify and
characterize the putative FHF-1 NLS, we determined the subcellular
localization of deletion mutants of FHF-1 by immunostaining with
anti-FHF-1 antibodies, and of fusions between FHF-1 and
.beta.-galactosidase by X-gal staining. These experiments show that
the two clusters of arginines and lysines at amino acids 11-18 and
28-38 in FHF-1 make up the two basic regions of a bipartite NLS. In
the context of the FHF-1 protein, deletion of either cluster
produced a modest increase in the level of cytoplasmic FHF-1, but
left significant nuclear accumulation (constructs 2 and 3; FIG.
14), while deletion of both regions abolished nuclear accumulation
(construct 4; FIGS. 14 and 15). To further characterize the minimal
region required for import, the first 56 or the first 69 residues
of FHF-1 were fused to beta-galactosidase and were shown to contain
a functional NLS (constructs 5 and 6; FIGS. 14 and 15). Fusion of
FHF-1 amino acids 1-22 or 23-55 individually to beta-galactosidase
failed to direct nuclear localization, indicating a requirement for
both parts of the bipartite NLS or a requirement for sequences
distal to the first 55 residues. With beta-galactosidase fused to
the first 69 amino acids of FHF-1, the results of a deletion
analysis resembled those seen with intact FHF-1 (constructs 5, 9,
10, and 11; FIGS. 14 and 15). Finally, fusion of FHF-1 amino acids
11-38, containing only the two clusters of basic amino acids
separated by a 10 amino acid spacer, conferred nuclear
localization, although less efficiently than the larger segments
that contained this region, suggesting that amino acids 1-10 or
56-68, outside of the bipartite NLS consensus, play an ancillary
role in nuclear localization.
[0110] It is to be understood that, while the invention has been
described with reference to the above detailed description, the
foregoing description is intended to illustrate, but not to limit,
the scope of the invention. Other aspects, advantages, and
modifications of the invention are within the scope of the
following claims. All publications, patent applications, patents,
and other references mentioned herein are incorporated by reference
in their entirety.
Sequence CWU 1
1
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