U.S. patent application number 09/754531 was filed with the patent office on 2002-06-27 for tubby 2 polypeptides.
Invention is credited to Doe, Trudy Rachel, Gloger, Israel Simon, Hughes, Stephen A., Terrett, Jonathan Alexander, Testa, Tania Tamson.
Application Number | 20020082385 09/754531 |
Document ID | / |
Family ID | 26147634 |
Filed Date | 2002-06-27 |
United States Patent
Application |
20020082385 |
Kind Code |
A1 |
Terrett, Jonathan Alexander ;
et al. |
June 27, 2002 |
Tubby 2 polypeptides
Abstract
Tubby 2 polypeptides and polynucleotides and methods for
producing such polypeptides by recombinant techniques are
disclosed. Also disclosed are methods for utilizing Tubby 2
polypeptides and polynucleotides in the design of protocols for the
treatment of diabetes, obesity, and atherosclerosis, among others,
and diagnostic assays for such conditions.
Inventors: |
Terrett, Jonathan Alexander;
(Chelmsford, GB) ; Testa, Tania Tamson; (London,
GB) ; Gloger, Israel Simon; (London, GB) ;
Hughes, Stephen A.; (Welwyn Garden City, GB) ; Doe,
Trudy Rachel; (Ware, GB) |
Correspondence
Address: |
Ratner & Prestia
Suite 301
One Westlakes, Berwyn
P.O. Box 980
Valley Forge
PA
19482-0980
US
|
Family ID: |
26147634 |
Appl. No.: |
09/754531 |
Filed: |
January 4, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09754531 |
Jan 4, 2001 |
|
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08977865 |
Nov 25, 1997 |
|
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6187908 |
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Current U.S.
Class: |
530/350 ;
435/69.1; 536/23.5 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/47 20130101 |
Class at
Publication: |
530/350 ;
435/69.1; 536/23.5; 514/12; 514/44 |
International
Class: |
A61K 038/00; C07H
021/04; A61K 031/70; C12P 021/06; C07K 001/00; C07K 014/00; C07K
017/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 25, 1996 |
GB |
9624433.0 |
Oct 6, 1997 |
EP |
97307877.7 |
Claims
What is claimed is:
1. An isolated polypeptide comprising an amino acid sequence which
has at least 70% identity to the amino acid sequence of SEQ ID NO:2
over the entire length of SEQ ID NO:2.
2. An isolated polypeptide as claimed in claim 1 in which the amino
acid sequence has at least 80% identity.
3. An isolated polypeptide as claimed in claim 1 in which the amino
acid sequence has at least 90% identity.
4. The polypeptide as claimed in claim 1 which comprises the amino
acid sequence of SEQ ID NO:2.
5. The polypeptide of SEQ ID NO:2.
6. An isolated polynucleotide comprising a nucleotide sequence that
has at least 70% identity to a nucleotide sequence encoding the
polypeptide of SEQ ID NO:2 over the entire coding region; or a
nucleotide sequence complementary to said isolated
polynucleotide.
7. An isolated polynucleotide as claimed in claim 6 in which the
nucleotide sequence has at least 80% identity.
8. An isolated polynucleotide as claimed in claim 6 in which the
nucleotide sequence that has at least 90% identity.
9. An isolated polynucleotide which comprises the nucleotide
sequence contained in SEQ ID NO: 1 encoding the polypeptide of SEQ
ID N02; or a nucleotide sequence complementary to said isolated
polynucleotide.
10. An isolated polynucleotide which comprises a nucleotide
sequence which has at least 70% identitv to that of SEQ ID NO: 1
over the entire length of SEQ ID NO: 1; or a nucleotide sequence
complementary to said isolated polynucleotide.
11. An isolated polynucleotide as claimed in claim 10 in which the
nucleotide sequence which has at least 80% identity.
12. An isolated polynucleotide as claimed in claim 10 in which the
nucleotide sequence which has at least 90% identity.
13. The polynucleotide of claim 10 which is the polynucleotide of
SEQ ID NO: 1.
14. An expression system comprising a DNA or RNA molecule which is
capable of producing a polypeptide of claim 1 when said expression
system is present in a compatible host cell.
15. A host cell comprising the expression system of claim 14.
16. A process for producing a polypeptide comprising culturing a
host of claim 14 under conditions sufficient for the production of
said polypeptide and recovering the polypeptide from the
culture.
17. An antibody immunospecific for the polypeptide of claim 1.
18. A method for the treatment of a subject in need of enhanced
activity or expression of the polypeptide of claim 1 comprising:
(a) administering to the subject a therapeutically effective amount
of an agonist to said polypeptide; and/or (b) providing to the
subject an isolated polynucleotide comprising a nucleotide sequence
of claim 6; or a nucleotide sequence complementary to said
nucleotide sequence in a form so as to effect production of said
polypeptide activity in vivo.
19. A method for the treatment of a subject having need to inhibit
activity or expression of the polypeptide of claim 1 comprising:
(a) administering to the subject a therapeutically effective amount
of an antagonist to said polypeptide; and/or (b) administering to
the subject a nucleic acid molecule that inhibits the expression of
the nucleotide sequence encoding said polypeptide; and/or (c)
administering to the subject a therapeutically effective amount of
a polypeptide that competes with said polypeptide for its ligand,
substrate, or receptor.
20. A process for diagnosing a disease or a susceptibility to a
disease in a subject related to expression or activity of the
polypeptide of claim 1 in a subject comprising: (a) deerrnining the
presence or absence of a mutation in the nucleotide sequence
encoding said polypeptide in the genome of said subject; and/or (b)
analyzing for the presence or amount of said polypeptide expression
in a sample derived from said subject.
21. An agonist of the polypeptide of claim 1.
22. An antagonist of the polypeptide of claim 1.
23. A polypeptide characterised by the deduced amino acid sequence
of SEQ ID NO:4; or a fragment thereof.
24. A polypeptide which has the amino acid sequence of SEQ ID
NO:4
25. A polynucleotide which encodes a polypeptide characterised by
the deduced amino acid sequence of SEQ ID NO:4.
26. A polynucleotide comprising the partial DNA sequence given in
SEQ ID NO:3.
27. The polynucloetide which has the sequence given in SEQ ID NO:3.
Description
FIELD OF INVENTION
[0001] This invention relates to newly identified polynucleotides,
polypeptides encoded by them and to the use of such polynucleotides
and polypeptides, and to their production. More particularly, the
polynucleotides and polypeptides of the present invention relate to
the Tubby related polypeptides family, hereinafter referred to as
Tubby 2. The invention also relates to inhibiting or activating the
action of such polynucleotides and polypeptides.
BACKGROUND OF THE INVENTION
[0002] The mouse tubby gene has been shown to be mutated in the
so-called `Tubby` mouse model (Kleyn, PW et al (1996) Cell 85,
281-290; Noben-Trauth, K et al. (1996) Nature 380: 534-538). Mutant
mice exhibit weight gain from age 6 months and develop insulin
resistance associated with this weight gain. There is also evidence
of increased susceptibility to atherosclerosis and dyslipidemia in
these mutant mice. Kleyn et al. also describe the human orthologue
of the mouse tubby gene. It is suggested that changes in the tubby
polypeptide are relevant to human weight gain disorders. Kleyn et
al however provide no description of the biochemical or signalling
properties of the tubby protein.
[0003] International patent application, publication number WO
96/05861, relates to compositions and methods for the treatment of
bodyweight disorders and in particular identifies certain genes
which are used in these composition and methods.
[0004] There is a need for identification and characterization of
further members of the Tubby related polypeptides family which can
play a role in preventing, ameliorating or correcting dysfunctions
or diseases.
SUMMARY OF THE INVENTION
[0005] In one aspect, the invention relates to Tubby 2 polypeptides
and recombinant materials and methods for their production. Another
aspect of the invention relates to methods for using such Tubby 2
polypeptides and polynucleotides. Such uses include the treatment
of diabetes, obesity, and atherosclerosis, among others. In still
another aspect, the invention relates to methods to identify
agonists and antagonists using the materials provided by the
invention, and treating conditions associated with Tubby 2
imbalance with the identified compounds. Yet another aspect of the
invention relates to diagnostic assays for detecting diseases
associated with inappropriate Tubby 2 activity or levels.
DESCRIPTION OF THE INVENTION
[0006] Polypeptides of the Invention
[0007] In a first aspect, the present invention relates to Tubby 2
polypeptides and variants and fragments thereof. Such polypeptides
include isolated polypetides comprising an amino acid sequence
which has at least 70% identity, preferably at least 80% identity,
more preferably at least 90% identity, yet more preferably at least
95% identity, most preferably at least 97-99% identity, to that of
SEQ ID NO:2 over the entire length of SEQ ID NO:2. Such
polypeptides include those comprising the amino acid of SEQ ID
NO:2.
[0008] Tubby 2 polypeptides also include isolated polypeptides in
which the amino acid sequence has at least 70% identity, preferably
at least 80% identity, more preferably at least 90% identity, yet
more preferably at least 95% identity, most preferably at least
97-99% identity, to the amino acid sequence of SEQ ID NO:2 over the
entire length of SEQ ID NO:2. Such polypeptides include the
polypeptide of SEQ ID NO:2.
[0009] The Tubby 2 polypeptides may be in the form of the "mature"
protein or may be a part of a larger protein such as a fusion
protein. It is often advantageous to include an additional amino
acid sequence which contains secretory or leader sequences,
pro-sequences, sequences which aid in purification such as multiple
histidine residues, or an additional sequence for stability during
recombinant production.
[0010] Fragments of the Tubby 2 polypeptides are also included in
the invention. A fragment is a polypeptide having an amino acid
sequence that entirely is the same as part, but not all, of the
amino acid sequence of the aforementioned Tubby 2 polypeptides. As
with Tubby 2 polypeptides, fragments may be "free-standing," or
comprised within a larger polypeptide of which they form a part or
region, most preferably as a single continuous region.
Representative examples of polypeptide fragments of the invention,
include, for example, fragments from about amino acid number 1-20,
21-40, 41-60, 61-80, 81-100, and 101 to the end of Tubby 2
polypeptide. In this context "about" includes the particularly
recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 amino
acid at either extreme or at both extremes.
[0011] Preferred fragments include, for example, truncation
polypeptides having the amino acid sequence of Tubby 2
polypeptides, except for deletion of a continuous series of
residues that includes the amino terminus, or a continuous series
of residues that includes the carboxyl terminus or deletion of two
continuous series of residues, one including the amino terminus and
one including the carboxyl terminus. Also preferred are fragments
characterized by structural or functional attributes such as
fragments that comprise alpha-helix and alpha-helix forming
regions, beta-sheet and beta-sheet-forming regions, turn and
turn-forming regions, coil and coil-forming regions, hydrophilic
regions, hydrophobic regions, alpha amphipathic regions, beta
amphipathic regions, flexible regions, surface-forming regions,
substrate binding region, and high antigenic index regions. Other
preferred fragments are biologically active fragments. Biologically
active fragments are those that mediate Tubby 2 activity, including
those with a similar activity or an improved activity, or with a
decreased undesirable activity. Also included are those that are
antigenic or immunogenic in an animal, especially in a human.
[0012] Variants of the defined sequence and fragments also form
part of the present invention. Preferred variants are those that
vary from the referents by conservative amino acid
substitutions----i.e., those that substitute a residue with another
of like characteristics. Typical such substitutions are among Ala,
Val, Leu and Ile; among Ser and Thr; among the acidic residues Asp
and Glu; among Asn and Gln; and among the basic residues Lys and
Arg; or aromatic residues Phe and Tyr. Particularly preferred are
variants in which several, 5-10, 1-5, or 1-2 amino acids are
substituted, deleted, or added in any combination.
[0013] The Tubby 2 polypeptides of the invention can be prepared in
any suitable manner. Such polypeptides include isolated naturally
occurring polypeptides, recombinantly produced polypeptides,
synthetically produced polypeptides, or polypeptides produced by a
combination of these methods. Means for preparing such polypeptides
are well understood in the art.
[0014] Polynucleotides of the Invention
[0015] In a further aspect, the present invention relates to Tubby
2 polynucleotides. Such polynucleotides include isolated
polynucleotides comprising a nucleotide sequence which has at least
70% identity, preferably at least 80% identity, more preferably at
least 90% identity, yet more preferably at least 95% identity, to a
nucleotide sequence encoding the polypeptide of SEQ ID NO:2, over
the entire coding region. In this regard, polynucleotides which
have at least 97% identity are highly preferred, whilst those with
at least 98-99% identity are more highly preferred, and those with
at least 99% identity are most highly preferred. Such
polynucleotides include a polynucleotide comprising the nucleotide
sequence contained in SEQ ID NO: 1 encoding the polypeptide of SEQ
ID NO:2.
[0016] Tubby 2 polynucleotides further includes isolated
polynucleotides comprising a nucleotide sequence which has at least
70% identity, preferably at least 80% identity, more preferably at
least 90% identity, yet more preferably at least 95% identity, to
SEQ ID NO: I over the entire length of SEQ ID NO: 1. In this
regard, polynucleotides which have at least 97% identity are highly
preferred, whilst those with at least 98-99% identiy are more
highly preferred, and those with at least 99 %identity are most
highly preferred. Such polynucleotides include a polynucleotide
which is the polynucleotide of SEQ ID NO: 1.
[0017] The invention also provides polynucleotides which are
complementary to such polynucleotides.
[0018] Tubby 2 of the invention is structurally related to other
proteins of the Tubby related polypeptides family, as shown by the
results of sequencing the cDNA encoding human Tubby 2. The cDNA
sequence of SEQ ID NO: I contains an open reading frame (nucleotide
number I to 1170) encoding a polypeptide of 390 amino acids of SEQ
ID NO:2. Amino acid sequence of Table 1 (SEQ ID NO:2) has about 60%
identity (using BlastX) in 266 amino acid residues with Tubby
[(P50586) Kleyn, PW, et al, Cell 1996 Apr 19;85(2):281-290].
Nucleotide sequence of Table I (SEQ ID NO:1) has about 60% identity
(using BlastN) in 371 nucleotide residues with Mus musculus tubby
candidate (tub) mRNA [(U52824) Kleyn, PW, et al, Cell 1996 Apr
19;85(2):281- 290 ].There is found to be significant conservation
of the dibasic potential protease cleavage sequences between tubby
and the Tubby 2 polypeptide (amino acids 302 and 383, Kleyn et al)
which indicate that the Tubby 2 polypeptide could be cleaved in
vivo into smaller active peptide(s). There is however lower
similarity towards the 5' end (amino acids 167 - 202, Kleyn at
al.), suggesting that there may be important functional differences
between tubby and the Tubby 2 polypeptide.
1Table 1.sup.a 1 ATGAAGATGC GACaGGCTAA GCTGGATTAT CAGAGGCTAC
TACTTGAGAA 51 GAGGCAAAGG AAAAAGCGCC TTGAGCCATT TATGGTGCAG
CCCAATCCAG 101 AAGCCAGGCT ACGTCGGGCA AAGCcAAGGG CCAGTGATGA
GCAGACTCCC 151 TTGGTGAACT GTCATACTCC CCACAGCAAT GTCATCTTAC
ATGGTATTGA 201 TGGTCCAGCT GCTGTCCTGA AACCAGACGA AGTTCATGCT
CCATCAGTAA 251 GCTCcTCTGT TGTGGAAGAA GATGCTGAAA ACACCGTGGA
TaCTGCTTCC 301 AAGCCAGGAC TTCAGGAGCG TCTCCAAAAG CATGATATCT
CTGAAAGTGT 351 GAACTTCGAT GAgGAGACTG ATGGAATATC CCAGTCAGCA
TGTTTAGAAA 401 GACCCAATTC TGCATCAAGC CAGAATTCAA CCGATACAGG
CACTTCCGGT 451 TCTGCTACTG CCGCCCAACC AGCTGATAAC cTCCTGGGAG
ACATAGACGA 501 CCTGGAGGAC TTTGTGTATA GTCCTGCCCc TCAAGGTGTC
ACAGTAAGAT 551 GTCGGATAAT CCGGGATAAA AGGGGAATGG ATCGGGGTCT
CTTCCCCACC 601 TACTATATGT ACTTGGAAAA AGAAGAAAAT CAGAAGATAT
TTCTTCTTGC 651 AGCTAGAAAG CGGAAAAAGA GCAAAACAGC CAACTACcTT
ATcTCCATtG 701 ATCCAGTTGA TTTATcTCGT GAAGGAGAAA GTTATGTCGG
CAAGCTTAGA 751 TCCAACCTCA TGGGGACCAA GTTTACAGTT TATGACCGTG
GCATCTGCCC 801 CATGAAGGGC CGGGGTTTGG TAGGAGCGGC CCACACCCGG
CAGGAGCTGG 851 CTGCCATCTC CTATGTGAGT GCTGCTTTCC CAGGGCCGCT
GCCTGCCCTC 901 CTGGTGTCCT GCTGGCACTT TTCACCTAGT GTCGCTGAAG
AACTCCCCTC 951 CcAAGCTTGT TTCTATTTCT GTGATTTCTG TTGCTGTACC
ATTTTCTCCA 1001 TGTATTTGAG TTTTAGTTAT TTGAATTGCC AAGTTCAATT
ATTTTTCACT 1051 CTCAGAACAT TTCTTCCCTT ATTTCCTTTT CTTTTTTCCT
GCTGCCACTT 1101 AATTCAGACC TTTACTTCTT ACCCAGTGGC CAAAGGTTAC
AATAAATTAA 1151 AGCTGATCTT TTTTTGCTTT TAATCTTTTC .sup.aA nucleotide
sequence of a human Tubby 2 (SEQ ID NO: 1) Table 2.sup.b 1
MKMRQAKLDY QRLLLEKRQR KKRLEPFMVQ PNPEARLRRA KPRASDEQTP 51
LVNCHTPHSN VILHGIDGPA AVLKPDEVHA PSVSSSVVEE DAENTVDTAS 101
KPGLQERLQK HDISESVNFD EETDGISQSA CLERPNSASS QNSTDTGTSG 151
SATAAQPADN LLGDIDDLED FVYSPAPQGV TVRCRIIRDK RGMDRGLFPT 201
YYMYLEKEEN QKIFLLAARK RKKSKTANYL ISIDPVDLSR EGESYVGKLR 251
SNLMGTKFTV YDRGICPMKG RGLVGAAHTR QELAAISYVS AAFPGPLPAL 301
LVSCWHFSPS VAEELPSQAC FYFCDFCCCT IFSMYLSFSY LNCQVQLFFT 351
LRTFLPLFPF LFSCCHLIQT FTSYPVAKGY NKLKLIFFCF .sup.BAn amino acid
sequence of a human Tubby 2 (SEQ ID NO: 2)
[0019] One polynucleotide of the present invention encoding Tubby 2
may be obtained using standard cloning and screening, from a cDNA
library derived from mRNA in cells of human Substantia Nigra using
the expressed sequence tag (EST) analysis (Adams, M.D., et al.
Science (1991) 252:1651-1656; Adams, M.D. et al., Nature, (1992)
355:632-634; Adams, M.D., et al., Nature (1995) 377 Supp:3-174).
Polynucleotides of the invention can also be obtained from natural
sources such as genomic DNA libraries or can be synthesized using
well known and commercialy available techniques.
[0020] The nucleotide sequence encoding Tubby 2 polypeptide of SEQ
ID NO:2 may be identical to the polypeptide encoding sequence
contained in Table 1 (nucleotide number I to 1170 of SEQ ID NO: 1),
or it may be a sequence, which as a result of the redundancy
(degeneracy) of the genetic code, also encodes the polypeptide of
SEQ ID NO:2.
[0021] When the polynucleotides of the invention are used for the
recombinant production of identical to the polypeptide, the
polynucleotide may include the coding sequence for the mature
polypeptide or a fragment thereof, by itself; the coding sequence
for the mature polypeptide or fragment in reading frame with other
coding sequences, such as those encoding a leader or secretory
sequence, a pre-, or pro- or prepro- protein sequence, or other
fusion peptide portions. For example, a marker sequence which
facilitates purification of the fused polypeptide can be encoded.
In certain preferred embodiments of this aspect of the invention,
the marker sequence is a hexa-histidine peptide, as provided.in the
pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc Natl
Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotide
may also contain non-coding 5' and 3' sequences, such as
transcribed, non-translated sequences, splicing and polyadenylation
signals, ribosome binding sites and sequences that stabilize
mRNA.
[0022] Further preferred embodiments are polynucleotides encoding
Tubby 2 variants comprising the amino acid sequence of Tubby 2
polypeptide of Table 2 (SEQ ID NO:2) in which several, 5-10, 1-5,
1-3, 1-2 or 1 amino acid residues are substituted, deleted or
added, in any combination.
[0023] The programme of work leading to the determination of SEQ ID
NO: 1 and 2 started with the preliminary identification of shorter
length gene fragments (ESTs). Accordingly, in a further aspect, the
present invention provides for a Tubby 2 characterised by the
deduced amino acid sequence of SEQ ID NO 4; or a fragment thereof,
and for polynucleotides which encode such polypeptides, in
particular, the polynucleotide comprising the partial DNA sequence
given in SEQ ID NO 3. In further aspects, the present invention
provides for the polynucleotide of SEQ ID NO: 3 encoding the amino
acid sequence of SEQ ID NO: 4, the polynucloetide which has the
sequence given in SEQ ID NO:3 and the polypeptide which has the
amino acid se4uence of SEQ ID NO:4. It will be readily appreciated
that the polynucleotide of SEQ ID NO:3 and the polypeptide of SEQ
ID NO:3 may be described as fragments of the polynucleotide of SEQ
ID NO: I and the polypeptide of SEQ ID NO:2, respectively. In
references herein to SEQ ID NO: 1, SEQ ID NO: 1 may be replaced by
SEQ ID NO:3 and in references herein to SEQ ID NO:2, SEQ ID NO:2
may be replaced by SEQ ID NO:4.
2Table 3.sup.C ggcacgagcttttttcccagaggctactacttgaga-
agaggcaaaggaaaaagcgccttgagccatttatggtgcagccc
aatccagaagccaggctacgtcgggcaaagcaagggccagtgatgagcagactcccttgggtgaactgtcata-
ctcccca cagcaatgtcatcttacatggtattgatggtccagctgctgtcctgaaac-
cagacgaaggttcatgctccatcagtaagc tctctgttgtggaagaagatgctgaaa-
acaccgtggatactgcttccaagccaggacttcaggagcgtctccaaaagcat
gatatctctgaaagtgtgaacttcgatgaggagactgatggaatatcccagtcagcatgtttagaaagaccca-
attctgc atcaagccagaattcaaccgatacaggcacttccggttctgctactgccg-
cccaaccagctgataacctcctgggagaca
tagacgacctggaggactttgtgtatagtcctgcccctcaaggtgtcacagtaagatgtcggataatccggga-
taaaagg ggaatggatcggggtctcttccccacctactatatgtacttggaaaaaga-
agaaaatcagaagatatttcttcttgcagc tagaaagcggaaaaagagcaaaacagc-
caactaccttatctccattgatccagttgatttatctcgtgaagganaaagtt atgtcgggg
.sup.cA partial nucleotide sequence of a human Tubby 2 (SEO ID NO:
3)
[0024]
3 Table 4.sup.d HELFSQRLLLEKRQRKKRLEPFMVQPNPEARL-
RRAKQGPVMSRLPWVNCHTP HSNVILHGIDGPAAVLKPDEGSCSISKLSVVEEDA-
ENTVDTASKPGLQERLQK HDISESVNFDEETDGISQSACLERPNSASSQNSTDTG-
TSGSATAAQPADNLLG DIDDLEDFVYSPAPQGVTVRCRIIRDKRGMDRGLFPTYY-
MYLEKEENQKIFLL AARKRKKSKTANYLISIDPVDLSREGXSYVG .sup.DA partial
amino acid sequence of a human Tubby 2 (SEQ ID NO: 4)
[0025] Whilst the polynucleotide of SEQID NO:3 does not include a
full open reading frame, a full length clone can readily be
obtained by methods well known to persons skilled in the art. Among
the alternative methods of choice, the following examples shuold be
mentioned: 1) The human library type cDNA library can either be
rescreened with a probe from the 5' end of the already cloned
sequence, 2) Anchor-PCR or RACE (Rapid Amplificaiton of cDNA Ends)
(Kriangkum et al., Nucleic Acids Res. 20 (1992) 3793 - 3794: Troutt
et al., Proc. Natl Acad. Sci., USA 89 (1992), 9823 -9825)
methodoloy can be used to clone the remaining 5' seqeuences from
Tissue Type RNA.3). The remaining 5' part can be isolated from
human genomic libraries, and DNA fragments considred to represent
introns can be identified on homology to the cDNA of the rat
receptor and deleted by mutagenesis.
[0026] After cloning of the 5' end for the open reading frame, this
part of the cDNA can be fused to the remaining 3' part of the human
T2 polypeptide cDNA by the use of PCR or through fusion at
appropriate restriction enzyme recognition seqeuences identied in
both the 5' and the 3' parts.
[0027] The cDNA encoding the full length open reading frame can be
cloned in suitable mammalian expression vectors and transfected
into mammalian cell lines for expression. Examples of such suitable
cell lines are the CHO and CHL cells, but other mammalian cells
will also express receptors o this type.
[0028] The present invention further relates to polynucleotides
that hybridize to the herein above-described sequences. In this
regard, the present invention especially relates to polynucleotides
which hybridize under stringent conditions to the herein
above-described polynucleotides. As herein used, the term
"stringent conditions" means hybridization will occur only if there
is at least 80%, and preferably at least 90%, and more preferably
at least 95%, yet even more preferably 97-99% identity between the
sequences.
[0029] Polynucleotides of the invention, which are identical or
sufficiently identical to a nucleotide sequence contained in SEQ ID
NO: I or a fragment thereof (including that of SEQ ID NO:3), may be
used as hybridization probes for cDNA and genomic DNA, to isolate
full-length cDNAs and genomic clones encoding Tubby 2 polypeptide
and to isolate cDNA and genomic clones of other genes (including
genes encoding homologs and orthologs from species other than
human) that have a high sequence similarity to the Tubby 2 gene.
Such hybridization techniques are known to those of skill in the
art. Typically these nucleotide sequences are 80% identical,
preferably 90% identical, more preferably 95% identical to that of
the referent. The probes generally will comprise at least 15
nucleotides. Preferably, such probes will have at least 30
nucleotides and may have at least 50 nucleotides. Particularly
preferred probes will range between 30 and 50 nucleotides.
[0030] In one embodiment, to obtain a polynucleotide encoding Tubby
2 polypeptide, including homologs and orthologs from species other
than human, comprises the steps of screening an appropriate library
under stingent hybridization conditions with a labeled probe having
the SEQ ID NO: I or a fragment thereof (including that of SEQ ID
NO: 3), and isolating full-length cDNA and genomic clones
containing said polynucleotide sequence. Such hybridization
techniques are well known to those of skill in the art. Thus in
another aspect, Tubby 2 polynucleotides of the present invention
further include a nucleotide sequence comprising a nucleotide
sequence that hybridize under stringent condition to a nucleotide
sequence having SEQ ID NO: 1 or a fragment thereof (including that
of SEQ ID NO:3). Also included with Tubby 2 polypeptides are
polypeptide comprising amnino acid sequence encoded by nucleotide
sequence obtained by the above hybridization condition. Stringent
hybridization conditions are as defined above or, alternatively,
conditions under overnight incubation at 42.degree. C. in a
solution comprising: 50% formamide, 5x SSC (150mM NaC1, 15mM
trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Derthardt's
solution, 10 % dextran sulfate, and 20 microgram/ml denatured,
sheared salmon sperm DNA, followed by washing the filters in 0.1x
SSC at about 65.degree. C.
[0031] The polynucleotides and polypeptides of the present
invention may be employed as research reagents and materials for
discovery of treatments and diagnostics to animal and human
disease.
[0032] Vectors, Host Cells, Expression
[0033] The present invention also relates to vectors which comprise
a polynucleotide or polynucleotides of the present invention, and
host cells which are genetically engineered with vectors of the
invention and to the production of polypeptides of the invention by
recombinant techniques. Cell-free translation systems can also be
employed to produce such proteins using RNAs derived from the DNA
constructs of the present invention.
[0034] For recombinant production, host cells can be genetically
engineered to incorporate expression systems or portions thereof
for polynucleotides of the present invention. Introduction of
polynucleotides into host cells can be effected by methods
described in many standard laboratory manuals, such as Davis et
al., BASIC METHODS IN MOLECULAR BIOLOGY(1986) and Sambrook et al.,
MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989) such as calcium
phosphate transfection, DEAE-dextran mediated transfection,
transvection, microinjection, cationic lipid-mediated transfection,
electroporation, transduction, scrape loading, ballistic
introduction or infection.
[0035] Representative examples of appropriate hosts include
bacterial cells, such as streptococci, staphylococci, E. coli,
Streptomyces and Bacillus subtilis cells; fungal cells, such as
yeast cells and Aspergillus cells; insect cells such as Drosophila
S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C
127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant
cells.
[0036] A great variety of expression systems can be used. Such
systems include, among others, chromosomal, episomal and
virus-derived systems, e.g., vectors derived from bacterial
plasmids, from bacteriophage, from transposons, from yeast
episomes, from insertion elements, from yeast chromosomal elements,
from viruses such as baculoviruses, papova viruses, such as SV40,
vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies
viruses and retroviruses, and vectors derived from combinations
thereof, such as those derived from plasmid and bacteriophage
genetic elements, such as cosmids and phagemids. The expression
systems may contain control regions that regulate as well as
engender expression. Generally, any system or vector suitable to
maintain, propagate or express polynucleotides to produce a
polypeptide in a host may be used. The appropriate nucleotide
sequence may be inserted into an expression system by any of a
variety of well-known and routine techniques, such as, for example,
those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY
MANUAL (supra).
[0037] For secretion of the translated protein into the lumen of
the endoplasmic reticulum, into the periplasmic space or into the
extracellular environment, appropriate secretion signals may be
incorporated into the desired polypeptide. These signals may be
endogenous to the polypeptide or they may be heterologous
signals.
[0038] If the Tubby 2 polypeptide is to be expressed for use in
screening assays, generally, it is preferred that the polypeptide
be produced at the surface of the cell. In this event, the cells
may be harvested prior to use in the screening assay. If Tubby 2
polypeptide is secreted into the medium, the medium can be
recovered in order to recover and purify the polypeptide; if
produced intracellularly, the cells must first be lysed before the
polypeptide is recovered. Tubby 2 polypeptides can be recovered and
purified from recombinant cell cultures by well-known methods
including ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. Most preferably, high
performance liquid chromatography is employed for purification.
Well known techniques for refolding proteins may be employed to
regenerate active conformation when the polypeptide is denatured
during isolation and or purification.
[0039] Diagnostic Assays
[0040] This invention also relates to the use of Tubby 2
polynucleotides for use as diagnostic reagents. Detection of a
mutated form of Tubby 2 gene associated with a dysfunction will
provide a diagnostic tool that can add to or define a diagnosis of
a disease or susceptibility to a disease which results from
under-expression, over-expression or altered expression of Tubby 2.
Individuals carrying mutations in the Tubby 2 gene may be detected
at the DNA level by a variety of techniques.
[0041] Nucleic acids for diagnosis may be obtained from a subject's
cells, such as from blood, urine, saliva, tissue biopsy or autopsy
material. The genomic DNA may be used directly for detection or may
be amplified enzymatically by using PCR or other amplification
techniques prior to analysis. RNA or cDNA may also be used in
similar fashion. Deletions and insertions can be detected by a
change in size of the amplified product in comparison to the normal
genotype. Point mutations can be identified by hybridizing
amplified DNA to labeled Tubby 2 nucleotide sequences. Perfectly
matched sequences can be distinguished from mismatched duplexes by
RNase digestion or by differences in melting temperatures. DNA
sequence differences may also be detected by alterations in
electrophoretic mobility of DNA fragments in gels, with or without
denaturing agents, or by direct DNA sequencing. See, e.g., Myers et
al., Science (1985) 230:1242. Sequence changes at specific
locations may also be revealed by nuclease protection assays, such
as RNase and SI protection or the chemical cleavage method. See
Cotton et al., Proc Natl Acad Sci USA (1985) 85: 4397-4401. In
another embodiment, an array of oligonucleotides probes comprising
Tubby 2 nucleotide sequence or fragments thereof can be constructed
to conduct efficient screening of e.g., genetic mutations. Array
technology methods are well known and have general applicability
and can be used to address a variety of questions in molecular
genetics including gene expression, genetic linkage, and genetic
variability. (See for example: M.Chee et al., Science, Vol 274, pp
610-613 (1996)).
[0042] The diagnostic assays offer a process for diagnosing or
determining a susceptibility to diabetes, obesity, and
atherosclerosis through detection of mutation in the Tubby 2 gene
by the methods described.
[0043] In addition, diabetes, obesity, and atherosclerosis can be
diagnosed by methods comprising determining from a sample derived
from a subject an abnormally decreased or increased level of Tubby
2 polypeptide or Tubby 2 mRNA. Decreased or increased expression
can be measured at the RNA level using any of the methods well
known in the art for the quantitation of polynucleotides, such as,
for example, PCR, RT-PCR, RNase protection, Northern blotting and
other hybridization methods. Assay techniques that can be used to
determine levels of a protein, such as an Tubby 2 polypeptide, in a
sample derived from a host are well-known to those of skill in the
art. Such assay methods include radioimmunoassays,
competitive-binding assays, Western Blot analysis and ELISA
assays.
[0044] Thus in another aspect, the present invention relates to a
diagonostic kit for a disease or suspectability to a disease,
particularly diabetes, obesity, and atherosclerosis, which
comprises:
[0045] (a) a Tubby 2 polynucleotide, preferably the nucleotide
sequence of SEQ ID NO: 1, or a fragment thereof;
[0046] (b) a nucleotide sequence complementary to that of (a);
[0047] (c) a Tubby 2 polypeptide, preferably the polypeptide of SEQ
ID NO: 2, or a fragment thereof; or
[0048] (d) an antibody to a Tubby 2 polypeptide, preferably to the
polypeptide of SEQ ID NO: 2.
[0049] It will be appreciated that in any such kit, (a), (b), (c)
or (d) may comprise a substantial component.
[0050] Chromosome Assays
[0051] The nucleotide sequences of the present invention are also
valuable for chromosome identification. The sequence is
specifically targeted to and can hybridize with a particular
location on an individual human chromosome. The mapping of relevant
sequences to chromosomes according to the present invention is an
important first step in correlating those sequences with gene
associated disease. Once a sequence has been mapped to a precise
chromosomal location, the physical position of the sequence on the
chromosome can be correlated with genetic map data. Such data are
found, for example, in V. McKusick, Mendelian Inheritance in Man
(available on line through Johns Hopkins University Welch Medical
Library). The relationship between genes and diseases that have
been mapped to the same chromosomal region are then identified
through linkage analysis (coinheritance of physically adjacent
genes). The differences in the cDNA or genomic sequence between
affected and unaffected individuals can also be determined. If a
mutation is observed in some or all of the affected individuals but
not in any normal individuals, then the mutation is likely to be
the causative agent of the disease.
[0052] The Tubby 2 gene has been mapped to chromosome 12p 13.3.
[0053] Antibodies
[0054] The polypeptides of the invention or their fragments or
analogs thereof, or cells expressing them can also be used as
immunogens to produce antibodies immunospecific for the Tubby 2
polypeptides. The term "immunospecific" means that the antibodies
have substantialI greater affinity for the polypeptides of the
invention than their affinity for other related polypeptides in the
prior art.
[0055] Antibodies generated against the Tubby 2 polypeptides can be
obtained by administering the polypeptides or epitope-bearing
fragments, analogs or cells to an animal, preferably a nonhuman,
using routine protocols. For preparation of monoclonal antibodies,
any technique which provides antibodies produced by continuous cell
line cultures can be used. Examples include the hybridoma technique
(Kohler, G. and Milstein, C., Nature (1975) 256:495-497), the
trioma technique, the human B-cell hybridoma technique (Kozbor et
al., Immunology Today (1983) 4:72) and the EBV-hybridoma technique
(Cole et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96,
Alan R. Liss, Inc., 1985).
[0056] Techniques for the production of single chain antibodies
(U.S. Pat. No. 4,946,778) can also be adapted to produce single
chain antibodies to polypeptides of this invention. Also,
transgenic mice, or other organisms including other mammals, may be
used to express humanized antibodies.
[0057] The above-described antibodies may be employed to isolate or
to identify clones expressing the polypeptide or to purify the
polypeptides by affinity chromatography.
[0058] Antibodies against Tubby 2 polypeptides may also be employed
to treat diabetes, obesity, and atherosclerosis, among others.
[0059] Vaccines
[0060] Another aspect of the invention relates to a method for
inducing an immunological response in a mammal which comprises
inoculating the mammal with Tubby 2 polypeptide, or a fragment
thereof, adequate to produce antibody and/or T cell immune response
to protect said animal from diabetes, obesity, and atherosclerosis,
among others. Yet another aspect of the invention relates to a
method of inducing immunological response in a mammal which
comprises, delivering Tubby 2 polypeptide via a vector directing
expression of Tubby 2 polynucleotide in vivo in order to induce
such an immunological response to produce antibody to protect said
animal from diseases.
[0061] Further aspect of the invention relates to an
immunological/vaccine formulation (composition) which, when
introduced into a mammalian host, induces an immunological response
in that mammal to a Tubby 2 polypeptide wherein the composition
comprises a Tubby 2 polypeptide or Tubby 2 gene. The vaccine
formulation may further comprise a suitable carrier. Since Tubby 2
polypeptide may be broken down in the stomach, it is preferably
administered parenterally (including subcutaneous, intramuscular,
intravenous, intradermal etc. injection). Formulations suitable for
parenteral administration include aqueous and non-aqueous sterile
injection solutions which may contain anti-oxidants, buffers,
bacteriostats and solutes which render the formulation instonic
with the blood of the recipient; and aqueous and non-aqueous
sterile suspensions which may include suspending agents or
thickening agents. The formulations may be presented in unit-dose
or multi-dose containers, for example, sealed ampoules and vials
and may be stored in a freeze-dried condition requiring only the
addition of the sterile liquid carrier immediately prior to use.
The vaccine formulation may also include adjuvant systems for
enhancing the immunogenicity of the formulation, such as oil-in
water systems and other systems known in the art. The dosage will
depend on the specific activity of the vaccine and can be readily
determined by routine experimentation.
[0062] Screening Assays
[0063] The Tubby 2 polypeptide of the present invention may be
employed in a screening process for compounds which activate
(agonists) or inhibit activation of (antagonists, or otherwise
called inhibitors) the Tubby 2 polypeptide of the present
invention. Thus, polypeptides of the invention may also be used to
assess identify agonist or antagonists from, for example, cells,
cell-free preparations, chemical libraries, and natural product
mixtures. These agonists or antagonists may be natural or modified
substrates, ligands, enzymes, receptors, etc., as the case may be,
of the polypeptide of the present invention; or may be structural
or functional mimetics of the polypeptide of the present invention.
See Coligan et al., Current Protocols in Immunology 1 (2):Chapter 5
(1991).
[0064] Tubby 2 polypeptides are responsible for many biological
functions, including many pathologies. Accordingly, it is desirous
to find compounds and drugs which stimulate Tubby 2 polypeptide on
the one hand and which can inhibit the function of Tubby 2
polypeptide on the other hand. In general, agonists are employed
for therapeutic and prophylactic purposes for such conditions as
Diabetes, obesity, atherosclerosis. Antagonists may be employed for
a variety of therapeutic and prophylactic purposes for such
conditions as diabetes, obesity, and atherosclerosis.
[0065] In general, such screening procedures may involve using
appropriate cells which express the Tubby 2 polypeptide or respond
to Tubby 2 polypeptide of the present invention. Such cells include
cells from mammals, yeast, Drosophila or E. coli. Cells which
express the Tubby 2 polypeptide (or cell membrane containing the
expressed polypeptide) or respond to Tubby 2 polypeptide are then
contacted with a test compound to observe binding, or stimulation
or inhibition of a functional response. The ability of the cells
which were contacted with the candidate compounds is compared with
the same cells which were not contacted for Tubby 2 activity.
[0066] The assays may simply test binding of a candidate compound
wherein adherence to the cells bearing the Tubby 2 polypeptide is
detected by means of a label directly or indirectly associated with
the candidate compound or in an assay involving competition with a
labeled competitor. Further, these assays may test whether the
candidate compound results in a signal generated by activation of
the Tubby 2 polypeptide, using detection systems appropriate to the
cells bearing the Tubby 2 polypeptide. Inhibitors of activation are
generally assayed in the presence of a known agonist and the effect
on activation by the agonist by the presence of the candidate
compound is observed.
[0067] Further, the assays may simply comprise the steps of mixing
a candidate compound with a solution containing a Tubby 2
polypeptide to form a mixture, measuring Tubby 2 activity in the
mixture, and comparing the Tubby 2 activity of the mixture to a
standard.
[0068] The Tubby 2 cDNA, protein and antibodies to the protein may
also be used to configure assays for detecting the effect of added
compounds on the production of Tubby 2 mRNA and protein in cells.
For example, an ELISA may be constructed for measuring secreted or
cell associated levels of Tubby 2 protein using monoclonal and
polyclonal antibodies by. standard methods known in the art, and
this can be used to discover agents which may inhibit or enhance
the production of Tubby 2 (also called antagonist or agonist,
respectively) from suitably manipulated cells or tissues.
[0069] The Tubby 2 protein may be used to identify membrane bound
or soluble receptors, if any, through standard receptor binding
techniques known in the art. These include, but are not limited to,
ligand binding and crosslinking assays in which the Tubby 2 is
labeled with a radioactive isotope (eg 125I), chemically modified
(eg biotinylated), or fused to a peptide sequence suitable for
detection or purification, and incubated with a source of the
putative receptor (cells, cell membranes, cell supernatants, tissue
extracts, bodily fluids). Other methods include biophysical
techniques such as surface plasmon resonance and spectroscopy. In
addition to being used for purification and cloning of the
receptor, these binding assays can be used to identify agonists and
antagonists of Tubby 2 which compete with the binding of Tubby 2 to
its receptors, if any. Standard methods for conducting screening
assays are well understood in the art.
[0070] Examples of potential Tubby 2 polypeptide antagonists
include antibodies or, in some cases, oligonucleotides or proteins
which are closely related to the ligands, substrates, enzymes,
receptors, etc., as the case may be, of the Tubby 2 polypeptide,
e.g., a fragment of the ligands, substrates, enzymes, receptors,
etc.; or small molecules which bind to the polypeptide of the
present invention but do not elicit a response, so that the
activity of the polypeptide is prevented.
[0071] Thus in another aspect, the present invention relates to a
screening kit for identifying agonists, antagonists, ligands,
receptors, substrates, enzymes, etc. for Tubby 2 polypeptides; or
compounds which decrease or enhance the production of Tubby 2
polypeptides, which comprises:
[0072] (a) a Tubby 2 polypeptide, preferably that of SEQ ID
NO:2;
[0073] (b) a recombinant cell expressing a Tubby 2 polypeptide,
preferably that of SEQ ID NO:2;
[0074] (c) a cell membrane expressing a Tubby 2 polypeptide;
preferably that of SEQ ID NO: 2; or
[0075] (d) antibody to a Tubby 2 polypeptide, preferably that of
SEQ ID NO: 2.
[0076] It will be appreciated that in any such kit, (a), (b), (c)
or (d) may comprise a substantial component.
[0077] Prophylactic and Therapeutic Methods
[0078] This invention provides methods of treating abnormal
conditions such as, Diabetes, obesity, atherosclerosis, related to
both an excess of and insufficient amounts of Tubby 2 polypeptide
activity.
[0079] If the activity of Tubby 2 polypeptide is in excess, several
approaches are available. One approach comprises administering to a
subject an inhibitor compound (antagonist) as hereinabove described
along with a pharmaceutically acceptable carrier in an amount
effective to inhibit the function of the Tubby 2 polypeptide, such
as, for example, by blocking the binding of ligands, substrates,
enzymes, receptors, etc., or by inhibiting a second signal, and
thereby alleviating the abnormal condition. In another approach,
soluble forms of Tubby 2 polypeptides still capable of binding the
ligand, substrate, enzymes, receptors, etc. in competition with
endogenous Tubby 2 polypeptide may be administered. Typical
embodiments of such competitors comprise fragments of the Tubby 2
polypeptide.
[0080] In another approach, soluble forms of Tubby 2 polypeptides
still capable of binding the ligand in competition with endogenous
Tubby 2 polypeptide may be administered. Typical embodiments of
such competitors comprise fragments of the Tubby 2 polypeptide.
[0081] In still another approach, expression of the gene encoding
endogenous Tubby 2 polypeptide can be inhibited using expression
blocking techniques. Known such techniques involve the use of
antisense sequences, either internally generated or separately
administered. See, for example, O'Connor,J Neurochem (1991) 56:560
in Oligodeoxynucleotides as Antisense Inhibitors of Gene
Expression, CRC Press, Boca Raton, FL (1988). Alternatively,
oligonucleotides which form triple helices with the gene can be
supplied. See, for example, Lee etal., Nucleic Acids Res (1979)
6:3073; Cooney etal., Science (1988) 241:456; Dervan etal., Science
(1991)251:1360. These oligomers can be administeredperse or the
relevant oligomers can be expressed in vivo.
[0082] For treating abnormal conditions related to an
under-expression of Tubby 2 and its activity, several approaches
are also available. One approach comprises administering to a
subject a therapeutically effective amount of a compound which
activates Tubby 2 polypeptide, i.e., an agonist as described above,
in combination with a pharmaceutically acceptable carrier, to
thereby alleviate the abnormal condition. Alternatively, gene
therapy may be employed to effect the endogenous production of
Tubby 2 by the relevant cells in the subject. For example, a
polynucleotide of the invention may be engineered for expression in
a replication defective retroviral vector, as discussed above. The
retroviral expression construct may then be isolated and introduced
into a packaging cell transduced with a retroviral plasmid vector
containing RNA encoding a polypeptide of the present invention such
that the packaging cell now produces infectious viral particles
containing the gene of interest. These producer cells may be
administered to a subject for engineering cells in vivo and
expression of the polypeptide in vivo. For overview of gene
therapy, see Chapter 20, Gene Therapy and other Molecular
Genetic-based Therapeutic Approaches, (and references cited
therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS
Scientific Publishers Ltd (1996). Another approach is to administer
a therapeutic amount of Tubby 2 polypeptides in combination with a
suitable pharmaceutical carrier.
[0083] Formulation and Administration
[0084] Peptides, such as the soluble form of Tubby 2 polypeptides,
and agonists and antagonist peptides or small molecules, may be
formulated in combination with a suitable pharmaceutical carrier.
Such formulations comprise a therapeutically effective amount of
the polypeptide or compound, and a pharmaceutically acceptable
carrier or excipient. Such carriers include but are not limited to,
saline, buffered saline, dextrose, water, glycerol, ethanol, and
combinations thereof. Formulation should suit the mode of
administration, and is well within the skill of the art. The
invention further relates to pharmaceutical packs and kits
comprising one or more containers filled with one or more of the
ingredients of the aforementioned compositions of the
invention.
[0085] Polypeptides and other compounds of the present invention
may be employed alone or in conjunction with other compounds, such
as therapeutic compounds.
[0086] Preferred forms of systemic administration of the
pharmaceutical compositions include injection, typically by
intravenous injection. Other injection routes, such as
subcutaneous, intramuscular, or intraperitoneal, can be used.
Alternative means for systemic administration include transmucosal
and transdermal administration using penetrants such as bile salts
or fusidic acids or other detergents. In addition, if properly
formulated in enteric or encapsulated formulations, oral
administration may also be possible. Administration of these
compounds may also be topical and/or localized, in the form of
salves, pastes, gels and the like.
[0087] The dosage range required depends on the choice of peptide,
the route of administration, the nature of the formulation, the
nature of the subject's condition, and the judgment of the
attending practitioner. Suitable dosages, however, are in the range
of 0.1 - 100 .mu.glkg of subject. Wide variations in the needed
dosage, however, are to be expected in view of the variety of
compounds available and the differing efficiencies of various
routes of administration. For example, oral administration would be
expected to require higher dosages than administration by
intravenous injection. Variations in these dosage levels can be
adjusted using standard empirical routines for optimization, as is
well understood in the art.
[0088] Polypeptides used in treatment can also be generated
endogenously in the subject, in treatment modalities often referred
to as "gene therapy" as described above. Thus, for example, cells
from a subject may be engineered with a polynucleotide, such as a
DNA or RNA, to encode a polypeptide ex vivo, and for example, by
the use of a retroviral plasmid vector. The cells are then
introduced into the subject.
[0089] This invention also provides a transgenic non-human animal
comprising a T2 polynucleotide encoding a T2 polypeptide. Also
provided are methods for use of said transgenic animals as models
for mutation and SAR (structure/activity relationship) evaluation
as well as in drug screens. Definitions The following definitions
are provided to facilitate understanding of certain terms used
frequently hereinbefore.
[0090] "Tubby 2 activity or Tubby 2 polypeptide activity" or
"biological activity of the Tubby 2 or Tubby 2 polypeptide" refers
to the metabolic or physiologic function of said Tubby 2 including
similar activities or improved activities or these activities with
decreased undesirable side-effects. Also included are antigenic and
immunogenic activities of said Tubby 2.
[0091] "Tubby 2 gene" refers to a polynucleotide having the
nucleotide sequence set forth in SEQ ID NO: 1 or allelic variants
thereof and/or their complements.
[0092] "Antibodies" as used herein includes polyclonal and
monoclonal antibodies, chimeric, single chain, and humanized
antibodies, as well as Fab fragments, including the products of an
Fab or other immunoglobulin expression library.
[0093] "Isolated" means altered "by the hand of man" from the
natural state. If an "isolated" composition or substance occurs in
nature, it has been changed or removed from its original
environment, or both. For example, a polynucleotide or a
polypeptide naturally present irra living animal is not "isolated,"
but the same polynucleotide or polypeptide separated from the
coexisting materials of its natural state is "isolated", as the
term is employed herein.
[0094] "Polynucleotide" generally refers to any polyribonucleotide
or polydeoxribonucleotide, which may be unmodified RNA or DNA or
modified RNA or DNA. "Polynucleotides" include, without limitation
single- and double-stranded DNA, DNA that is a mixture of single-
and double-stranded regions, single- and double-stranded RNA, and
RNA that is mixture of single- and double-stranded regions, hybrid
molecules comprising DNA and RNA that may be single-stranded or,
more typically, double-stranded or a mixture of single- and
double-stranded regions. In addition, "polynucleotide" refers to
triple-stranded regions comprising RNA or DNA or both RNA and DNA.
The term polynucleotide also includes DNAs or RNAs containing one
or more modified bases and DNAs or RNAs with backbones modified for
stability or for other reasons. "Modified" bases include, for
example, tritylated bases and unusual bases such as inosine. A
variety of modifications has been made to DNA and RNA; thus,
"polynucleotide" embraces chemically, enzymatically or
metabolically modified forms of polynucleotides as typically found
in nature, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells. "Polynucleotide" also embraces
relatively short polynucleotides, often referred to as
oligonucleotides.
[0095] "Polypeptide" refers to any peptide or protein comprising
two or more amino acids joined to each other by peptide bonds or
modified peptide bonds, i.e., peptide isosteres. "Polypeptide"
refers to both short chains, commonly referred to as peptides,
oligopeptides or oligomers, and to longer chains, generally
referred to as proteins. Polypeptides may contain amino acids other
than the 20 gene-encoded amino acids. "Polypeptides" include amino
acid sequences modified either by natural processes, such as
posttranslational processing, or by chemical modification
techniques which are well known in the art. Such modifications are
well described in basic texts and in more detailed monographs, as
well as in a voluminous research literature. Modifications can
occur anywhere in a polypeptide, including the peptide backbone,
the amino acid side-chains and the amino or carboxyl termini. It
will be appreciated that the same type of modification may be
present in the same or varying degrees at several sites in a given
polypeptide. Also, a given polypeptide may contain many types of
modifications. Polypeptides may be branched as a result of
ubiquitination, and they may be cyclic, with or without branching.
Cyclic, branched and branched cyclic polypeptides may result from
posttranslation natural processes or may be made by synthetic
methods. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent cross-links, formation of
cystine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation,
proteolytic processing, phosphorylation, prenylation, racemization,
selenoylation, sulfation, transfer-RNA mediated addition of amino
acids to proteins such as arginylation, and ubiquitination. See,
for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd
Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993 and
Wold, F., Posttranslational Protein Modifications: Perspectives and
Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF
PROTEINS, B. C. Johnson, Ed., Academic Press, N. Y., 1983; Seifter
et aL, "Analysis for protein modifications and nonprotein
cofactors", Meth Enzymol (1990) 182:626-646 and Rattan et al.,
"Protein Synthesis: Posttranslational Modifications and Aging", Ann
N.Y. Acad Sci (1 992) 663:48-62. "Variant" as the term is used
herein, is a polynucleotide or polypeptide that differs from a
reference polynucleotide or polypeptide respectively, but retains
essential properties. A typical variant of a polynucleotide differs
in nucleotide sequence from another, reference polynucleotide.
Changes in the nucleotide sequence of the variant may or may not
alter the amino acid sequence of a polypeptide encoded by the
reference polynucleotide. Nucleotide changes may result in amino
acid substitutions, additions, deletions, fusions and truncations
in the polypeptide encoded by the reference sequence, as discussed
below. A typical variant of a polypeptide differs in amino acid
sequence from another, reference polypeptide. Generally,
differences are limited so that the sequences of the reference
polypeptide and the variant are closely similar overall and, in
many regions, identical. A variant and reference polypeptide may
differ in amino acid sequence by one or more substitutions,
additions, deletions in any combination. A substituted or inserted
amino acid residue may or may not be one encoded by the genetic
code. A variant of a polynucleotide or polypeptide may be a
naturally occurring such as an allelic variant, or it may be a
variant that is not known to occur naturally. Non-naturally
occurring variants of polynucleotides and polypeptides may be made
by mutagenesis techniques or by direct synthesis.
[0096] "Identity" is a measure of the identity of nucleotide
sequences or amino acid sequences. In general, the sequences are
aligned so that the highest order match is obtained. "Identity" per
se has an art-recognized meaning and can be calculated using
published techniques. See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY,
Lesk, A.M., ed., Oxford University Press, New York, 1988;
BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D.W., ed.,
Academic Press, N. Y., 1993; COMPUTER ANALYSIS OF SEQUENCE DATA,
PART I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, N.
J., 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G.,
Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER, Gribskov, M.
and Devereux, J., eds., M Stockton Press, N. Y., 1991). While there
exist a number of methods to measure identity between two
polynucleotide or polypeptide sequences, the term "identity" is
well known to skilled artisans (Carillo, H., and Lipton, D., SLAM J
Applied Math (1988) 48:1073). Methods commonly employed to
determine identity or similarity between two sequences include, but
are not limited to, those disclosed in Guide to Huge Computers,
Martin J. Bishop, ed., Academic Press, San Diego, 1994, and
Carillo, H., and Lipton, D., SLAM J Applied Math (1988) 48:1073.
Methods to determine identity and similarity are codified in
computer programs. Preferred computer program methods to determine
identity and similarity between two sequences include, but are not
limited to, GCS program package (Devereux, J., et al., Nucleic
Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul,
S.F. et al., J Molec Biol (1990) 215:403).
[0097] As an illustration, by a polynucleotide having a nucleotide
sequence having at least, for example, 95% "identity" to a
reference nucleotide sequence of SEQ ID NO: I is intended that the
nucleotide sequence of the polynucleotide is identical to the
reference sequence except that the polynucleotide sequence may
include up to five point mutations per each 100 nucleotides of the
reference nucleotide sequence of SEQ ID NO: 1. In other words, to
obtain a polynucleotide having a nucleotide sequence at least 95%
identical to a reference nucleotide sequence, up to 5% of the
nucleotides in the reference sequence may be deleted or substituted
with another nucleotide, or a number of nucleotides up to 5% of the
total nucleotides in the reference sequence may be inserted into
the reference sequence. These mutations of the reference sequence
may occur at the 5 or 3 terminal positions of the reference
nucleotide sequence or anywhere between those terminal positions,
interspersed either individually among nucleotides in the reference
sequence or in one or more contiguous groups within the reference
sequence. Similarly, by a polypeptide having an amino acid sequence
having at least, for example, 95% "identity" to a reference amino
acid sequence of SEQ ID NO:2 is intended that the amino acid
sequence of the polypeptide is identical to the reference sequence
except that the polypeptide sequence may include up to five amino
acid alterations per each 100 amino acids of the reference amino
acid of SEQ ID NO: 2. In other words, to obtain a polypeptide
having an amino acid sequence at least 95% identical to a reference
amino acid sequence, up to 5% of the amino acid residues in the
reference sequence may be deleted or substituted with another amino
acid, or a number of amino acids up to 5% of the total amino acid
residues in the reference sequence may be inserted into the
reference sequence. These alterations of the reference sequence may
occur at the amino or carboxy terminal positions of the reference
amino acid sequence or anywhere between those terminal positions,
interspersed either individually among residues in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0098] All publications, including but not limited to patents and
patent applications, cited in this specification are herein
incorporated by reference as if each individual publication were
specifically and individually indicated to be incorporated by
reference herein as though fully set forth.
Example
An EST sequence (Human Genome Sciences, HGS 160391 1) was
identified as having homology with the published human and mouse
tubby gene sequences (Kleyn et al 1996). Further ESTs with the same
project ID (HSXAK37) from a substantia nigra cDNA library were
identified as derived from the more 5' region of the novel Tubby 2
gene. Oligos were designed in each of the non-overlapping EST
sequences and PCR between these non-overlapping sequences was
performed and the resulting products cloned and sequenced. The set
of HGS plus SB generated sequences were combined to create the
polynucleotide (SEQ ID NO 3) and the deduced polypeptide (SEQ ID NO
4).
[0099] The template for this PCR was Clontech Foetal Brain cDNA in
lambda gt 11 vector (Clontech Laboratories inc., 4030 Fabian Way,
Palo Alto, CA 94303-40607, USA). PCR reaction conditions were
94.degree. C. for 45 seconds, 60.degree. C. for 45 seconds,
72.degree. C. for 2 minutes, 35 cycles in an MJ PTC-100 thermal
cycler.
[0100] Expression profiles were determined using the same primers
and conditions as above on the Clontech Quickscreen panel.
[0101] The Chromosomal localisation of the Tubby 2 gene was
determined using oligos Tb3 and Tb4 on the GeneBridge 4 radiation
hybrid panel (Research Genetics, USA) using the conditions:
94.degree. C. for 40 seconds, 65.degree. C. for 30 seconds, 35
cycles on the MJ PTC-100 thermal cycler. This has positioned the
Tubby 2 polypeptide on chromosome 12P 13.
Sequence CWU 1
1
4 1 1180 DNA HOMO SAPIENS 1 atgaagatgc gacaggctaa gctggattat
cagaggctac tacttgagaa gaggcaaagg 60 aaaaagcgcc ttgagccatt
tatggtgcag cccaatccag aagccaggct acgtcgggca 120 aagccaaggg
ccagtgatga gcagactccc ttggtgaact gtcatactcc ccacagcaat 180
gtcatcttac atggtattga tggtccagct gctgtcctga aaccagacga agttcatgct
240 ccatcagtaa gctcctctgt tgtggaagaa gatgctgaaa acaccgtgga
tactgcttcc 300 aagccaggac ttcaggagcg tctccaaaag catgatatct
ctgaaagtgt gaacttcgat 360 gaggagactg atggaatatc ccagtcagca
tgtttagaaa gacccaattc tgcatcaagc 420 cagaattcaa ccgatacagg
cacttccggt tctgctactg ccgcccaacc agctgataac 480 ctcctgggag
acatagacga cctggaggac tttgtgtata gtcctgcccc tcaaggtgtc 540
acagtaagat gtcggataat ccgggataaa aggggaatgg atcggggtct cttccccacc
600 tactatatgt acttggaaaa agaagaaaat cagaagatat ttcttcttgc
agctagaaag 660 cggaaaaaga gcaaaacagc caactacctt atctccattg
atccagttga tttatctcgt 720 gaaggagaaa gttatgtcgg caagcttaga
tccaacctca tggggaccaa gtttacagtt 780 tatgaccgtg gcatctgccc
catgaagggc cggggtttgg taggagcggc ccacacccgg 840 caggagctgg
ctgccatctc ctatgtgagt gctgctttcc cagggccgct gcctgccctc 900
ctggtgtcct gctggcactt ttcacctagt gtcgctgaag aactcccctc ccaagcttgt
960 ttctatttct gtgatttctg ttgctgtacc attttctcca tgtatttgag
ttttagttat 1020 ttgaattgcc aagttcaatt atttttcact ctcagaacat
ttcttccctt atttcctttt 1080 cttttttcct gctgccactt aattcagacc
tttacttctt acccagtggc caaaggttac 1140 aataaattaa agctgatctt
tttttgcttt taatcttttc 1180 2 390 PRT HOMO SAPIENS 2 Met Lys Met Arg
Gln Ala Lys Leu Asp Tyr Gln Arg Leu Leu Leu Glu 1 5 10 15 Lys Arg
Gln Arg Lys Lys Arg Leu Glu Pro Phe Met Val Gln Pro Asn 20 25 30
Pro Glu Ala Arg Leu Arg Arg Ala Lys Pro Arg Ala Ser Asp Glu Gln 35
40 45 Thr Pro Leu Val Asn Cys His Thr Pro His Ser Asn Val Ile Leu
His 50 55 60 Gly Ile Asp Gly Pro Ala Ala Val Leu Lys Pro Asp Glu
Val His Ala 65 70 75 80 Pro Ser Val Ser Ser Ser Val Val Glu Glu Asp
Ala Glu Asn Thr Val 85 90 95 Asp Thr Ala Ser Lys Pro Gly Leu Gln
Glu Arg Leu Gln Lys His Asp 100 105 110 Ile Ser Glu Ser Val Asn Phe
Asp Glu Glu Thr Asp Gly Ile Ser Gln 115 120 125 Ser Ala Cys Leu Glu
Arg Pro Asn Ser Ala Ser Ser Gln Asn Ser Thr 130 135 140 Asp Thr Gly
Thr Ser Gly Ser Ala Thr Ala Ala Gln Pro Ala Asp Asn 145 150 155 160
Leu Leu Gly Asp Ile Asp Asp Leu Glu Asp Phe Val Tyr Ser Pro Ala 165
170 175 Pro Gln Gly Val Thr Val Arg Cys Arg Ile Ile Arg Asp Lys Arg
Gly 180 185 190 Met Asp Arg Gly Leu Phe Pro Thr Tyr Tyr Met Tyr Leu
Glu Lys Glu 195 200 205 Glu Asn Gln Lys Ile Phe Leu Leu Ala Ala Arg
Lys Arg Lys Lys Ser 210 215 220 Lys Thr Ala Asn Tyr Leu Ile Ser Ile
Asp Pro Val Asp Leu Ser Arg 225 230 235 240 Glu Gly Glu Ser Tyr Val
Gly Lys Leu Arg Ser Asn Leu Met Gly Thr 245 250 255 Lys Phe Thr Val
Tyr Asp Arg Gly Ile Cys Pro Met Lys Gly Arg Gly 260 265 270 Leu Val
Gly Ala Ala His Thr Arg Gln Glu Leu Ala Ala Ile Ser Tyr 275 280 285
Val Ser Ala Ala Phe Pro Gly Pro Leu Pro Ala Leu Leu Val Ser Cys 290
295 300 Trp His Phe Ser Pro Ser Val Ala Glu Glu Leu Pro Ser Gln Ala
Cys 305 310 315 320 Phe Tyr Phe Cys Asp Phe Cys Cys Cys Thr Ile Phe
Ser Met Tyr Leu 325 330 335 Ser Phe Ser Tyr Leu Asn Cys Gln Val Gln
Leu Phe Phe Thr Leu Arg 340 345 350 Thr Phe Leu Pro Leu Phe Pro Phe
Leu Phe Ser Cys Cys His Leu Ile 355 360 365 Gln Thr Phe Thr Ser Tyr
Pro Val Ala Lys Gly Tyr Asn Lys Leu Lys 370 375 380 Leu Ile Phe Phe
Cys Phe 385 390 3 729 DNA HOMO SAPIENS UNSURE (714) 3 ggcacgagct
tttttcccag aggctactac ttgagaagag gcaaaggaaa aagcgccttg 60
agccatttat ggtgcagccc aatccagaag ccaggctacg tcgggcaaag caagggccag
120 tgatgagcag actcccttgg gtgaactgtc atactcccca cagcaatgtc
atcttacatg 180 gtattgatgg tccagctgct gtcctgaaac cagacgaagg
ttcatgctcc atcagtaagc 240 tctctgttgt ggaagaagat gctgaaaaca
ccgtggatac tgcttccaag ccaggacttc 300 aggagcgtct ccaaaagcat
gatatctctg aaagtgtgaa cttcgatgag gagactgatg 360 gaatatccca
gtcagcatgt ttagaaagac ccaattctgc atcaagccag aattcaaccg 420
atacaggcac ttccggttct gctactgccg cccaaccagc tgataacctc ctgggagaca
480 tagacgacct ggaggacttt gtgtatagtc ctgcccctca aggtgtcaca
gtaagatgtc 540 ggataatccg ggataaaagg ggaatggatc ggggtctctt
ccccacctac tatatgtact 600 tggaaaaaga agaaaatcag aagatatttc
ttcttgcagc tagaaagcgg aaaaagagca 660 aaacagccaa ctaccttatc
tccattgatc cagttgattt atctcgtgaa gganaaagtt 720 atgtcgggg 729 4 242
PRT HOMO SAPIENS UNSURE (238) 4 His Glu Leu Phe Ser Gln Arg Leu Leu
Leu Glu Lys Arg Gln Arg Lys 1 5 10 15 Lys Arg Leu Glu Pro Phe Met
Val Gln Pro Asn Pro Glu Ala Arg Leu 20 25 30 Arg Arg Ala Lys Gln
Gly Pro Val Met Ser Arg Leu Pro Trp Val Asn 35 40 45 Cys His Thr
Pro His Ser Asn Val Ile Leu His Gly Ile Asp Gly Pro 50 55 60 Ala
Ala Val Leu Lys Pro Asp Glu Gly Ser Cys Ser Ile Ser Lys Leu 65 70
75 80 Ser Val Val Glu Glu Asp Ala Glu Asn Thr Val Asp Thr Ala Ser
Lys 85 90 95 Pro Gly Leu Gln Glu Arg Leu Gln Lys His Asp Ile Ser
Glu Ser Val 100 105 110 Asn Phe Asp Glu Glu Thr Asp Gly Ile Ser Gln
Ser Ala Cys Leu Glu 115 120 125 Arg Pro Asn Ser Ala Ser Ser Gln Asn
Ser Thr Asp Thr Gly Thr Ser 130 135 140 Gly Ser Ala Thr Ala Ala Gln
Pro Ala Asp Asn Leu Leu Gly Asp Ile 145 150 155 160 Asp Asp Leu Glu
Asp Phe Val Tyr Ser Pro Ala Pro Gln Gly Val Thr 165 170 175 Val Arg
Cys Arg Ile Ile Arg Asp Lys Arg Gly Met Asp Arg Gly Leu 180 185 190
Phe Pro Thr Tyr Tyr Met Tyr Leu Glu Lys Glu Glu Asn Gln Lys Ile 195
200 205 Phe Leu Leu Ala Ala Arg Lys Arg Lys Lys Ser Lys Thr Ala Asn
Tyr 210 215 220 Leu Ile Ser Ile Asp Pro Val Asp Leu Ser Arg Glu Gly
Xaa Ser Tyr 225 230 235 240 Val Gly
* * * * *