Note: Descriptions are shown in the official language in which they were submitted.
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HUMAN DEUBIQUITINATING PROTEASE GENE ON CHROMOSOME 7
AND ITS MURINE ORTHOLOG
Background of the Invention
The role of ubiquitin in protein degradation was discovered and the main
enzymatic
reactions of this system elucidated in biochemical studies in a cell-free
system from
reticulocytes. In this system, proteins are targeted for degradation by
covalent ligation to
0 ubiquitin, a 76-amino-acid-residue protein. Briefly, ubiquitin-protein
ligation requires the
sequential action of three enzymes. The C-terminal Gly residue of ubiquitin is
activated in an
ATP-requiring step by a specific activating enzyme, El (Step 1). This step
consists of an
intermediate formation of ubiquitin adenylate, with the release of PP;,
followed by the binding
of ubiquitin to a Cys residue of El in a thiolester linkage, with the release
of AMP. Activated
ubiquitin is next transferred to an active site Cys residue of a ubiquitin-
carrier protein, E2
(Step 2). W the third step catalyzed by a ubiquitin-protein ligase or E3
enzyme, ubiquitin is
linked by its C-terminus in an amide isopeptide linkage to an -amino group of
the substrate
protein's Lys residues (Step 3).
Proteins ligated to polyubiquitin chains are usually degradedby the 26S
proteasome
complex that requires ATP hydrolysis for its action. The 26S proteasome is
formedby an
ATP-dependent assembly of a 20S proteasome, a complex that contains the
protease catalytic
sites, with 19S "cap" or regulatory complexes. The 19S complexes contain
several ATPase
subunits and other subunits that are presumably involved in the specific
action of the 26S
?5 proteasome on ubiquitinylatedproteins. The roles of ATP in the assembly of
the 26S
proteasome complex and in its proteolytic action are not understood. The
action of the 26S
proteasome presumably generates several types of products: free peptides,
short peptides still
linked to ubiquitin via their Lys residues, and polyubiquitin chains (Step 4).
The latter two
products are converted to free and reusable ubiquitin by the action of
ubiquitin-C-terminal
3o hydrolases or isopeptidases (Steps 5 and 6). Some isopeptidases may also
disassemble certain
ubiquitin-protein conjugates (Step 7) and thus prevent their proteolysis by
the 26S proteasome.
The latter type of isopeptidase action may have a correction function to
salvage incorrectly
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ubiquitinylated proteins or may have a regulatory role. Short peptides formed
by the above
processes can be further degraded to free amino acids by cytosolic peptidases
(Step 8).
Ubiquitin-mediated degradation of protein is involved in various biological
processes.
The selective and prograrmned degradation of cell-cycle regulatory proteins,
such as cyclins,
inhibitors of cyclin-dependent l~inases, and anaphase inhibitors are essential
events in cell-
cycle progression. Cell growth and proliferation are further controlled by
ubiquitin-mediated
degradation of tumor suppressors, protooncogenes, and components of signal
transduction
systems. The rapid degradation of numerous transcriptional regulators is
involved in a variety
of signal transduction processes and responses to environmental cues. The
ubiquitin system is
clearly involved in endocytosis and down-regulation of receptors and
transporters, as well as
in the degradation of resident or abnormal proteins in the endoplasmic
reticulum. There are
strong indications for roles of the ubiquitin system in development and
apoptosis, although the
target proteins involved in these cases have not been identified. Dysfunction
in several
l5 ubiquitin-mediated processes causes pathological conditions, including
malignant
transformation.
Our knowledge of different signals in proteins that mark them for
ubiquitinylation is
also limited. Recent reports indicate that many proteins are targeted for
degradation by
?0 phosphorylation. It was observed previously that many rapidly degraded
proteins contain
PEST elements, regions enriched in Pro, Glu, Ser, and Thr residues. More
recently, it was
pointed out that PEST elements are rich in S/TP sequences, which are minimum
consensus
phosphorylation sites for Cdks and some other protein kinases. Indeed, it now
appears that in
several (though certainly not all) instances, PEST elements contain
phosphorylation sites
25 necessary for degradation. Thus multiple phosphorylations within PEST
elements are required
for the ubiquitinylation and degradation of the yeast Gl cyclins Cln3 and
Cln2, as well as the
Gcn4 transcriptional activator. Otherproteins, such as the mammalian Gl
regulators cyclin E
and cyclin D 1, are targeted for ubiquitinylation by phosphorylation at
specific, single sites. In
the case of the IkBoc inhibitor of the NF-kB transcriptional regulator,
phosphorylation at two
3o specific sites, Ser-32 and Ser-36, is required for ubiquitin ligation. /3-
cateinin, which is
targeted for ubiquitin-mediated degradation by phosphorylation, has a sequence
motif similar
to that of llcBa around these phosphorylation sites. However, the homology in
phosphorylation patterns of these two proteins is not complete, because
phosphorylation of
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other sites of 13-cateun is also required for its degradation. Other proteins
targeted for
degradation by phosphorylation include the Cdk inhibitor Siclp and the STATl
transcription
factor. Though different patterns of phosphorylation target different proteins
for degradation, a
common feature appears to be that the initial regulatory event is carried out
by a protein
kinase, while the role of a ubiquitin ligase would be to recognize the
phosphorylated form of
the protein substrate. It further appears that different ubiquitin ligases
recognize different
phosphorylation patterns as well as additional motifs in the various protein
substrates.
However, the identity of such E3s is unknown, except for some PULC-type
ubiquitin ligases
that act on some phosphorylated cell-cycle regulators in the budding yeast.
The multiplicity of
l0 signals that target proteins for ubiquitin-mediated degradation (and of
ligases that have to
recognize such signals) is underscored by observations that the
phosphorylation of some
proteins actually prevents their degradation. Thus the phosphorylation of the
c-Mos
protooncogene on Ser3 and the multiple phosphorylations of c-Fos and c-Jun
protooncogenes
at multiple sites by MAP kinases suppress their ubiquitinylation and
degradation.
In addition to the families of enzymes involved in conjugation of ubiquitin, a
very
large family of deubiquitinating enzymes has recently been identified from
various organisms.
These enzymes have several possible functions. First, they may have peptidase
activity and
cleave the products of ubiquitin genes. Ubiquitin is encoded by two distinct
classes of genes.
2o One is a polyubiquitin gene, wluch encodes a linear polymer of ubiquitins
linked through
peptide bonds between the C-terminal Gly and N-terminal Met of contiguous
ubiquitin
molecules. Each copy of ubiquitin must be released by precise cleavage of the
peptide bond
between Gly-76-Met-1 of successive ubiquitinmoieties. The other class of
ubiquitin genes
encodes ubiquitin C-terminal extension proteins, which are peptide bond
fusions between the
C-terminal Gly of ubiquitin and N-terminal Met ofthe extension protein. To
date, the
extensions described are ribosomal proteins consisting of 52 or 76-~0 amino
acids. These
ubiquitin fusion proteins are processed to yield ubiquitin and the
corresponding C-terminal
extension proteins. Second, deubiquitinating enzymes may have isopeptidase
activities. When
a target protein is degraded, deubiquitinating enzymes can cleave the
polyubiquitin chain from
3o the target protein or its remnants. The polyubiquitin chain must also be
disassembledby
deubiquitinating enzymes during or after proteolysis by the 26 S proteasome,
regenerating free
monomeric ubiquitin. In this way, deubiquitinating enzymes can facilitate the
ability of the 26
S proteasome to degrade ubiquitinated proteins. Third, deubiquitinating
enzymes may
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hydrolyze ester, thiolester, and amide linkages to the carboxyl group of Gly-
76 of ubiquitin.
Such nonfunctional linlcages may arise from reactions between small
intracellular compounds
such as glutathione and the E1-, E2-, or E3-ubiquitin thiolester
intermediates. Fourth,
deubiquitinating enzymes may compete with the conjugating system by removing
ubiquitin
from protein substrates, thereby rescuing them from degradation or any other
function
mediated by ubiquitination. Thus generation of ubiquitin by deubiquitinating
enzymes from
the linear polyubiquitin and ubiquitin fusion proteins and from the branched
polyubiquitin
ligated to proteins should be essential for maintaining a sufficientpool of
free ubiquitin. Many
deubiquitinating enzymes exist, suggesting that these deubiquitinating enzymes
recognize
o distinct substrates and are therefore involved in specific cellular
processes. Although there is
recent evidence to support such specificity of these deubiquitinating enzymes,
the structure-
function relationships of these enzymes remain poorly studied.
Deubiquitinating enzymes can be divided broadly on the basis of sequence
homology
l5 into two classes, the ubiquitin-specific processing protease (UBP or USP,
also known as type
2 ubiquitin C-terminal hydrolase (type 2 UCH)) and the UCH, also known as type
1 UCH).
UCH (type 1 UCH) enzymes hydrolyze primarily C-terminal esters and amides of
ubiquitin
but may also cleave ubiquitin gene products and disassemble polyubiquitin
chains . They have
in common a 210-amino acid catalytic domain, with four highly conserved blocks
of
sequences that identify these enzymes. They contain two very conserved motifs,
the CYS and
HIS boxes. Mutagenesis studies revealed that the two boxes play important
roles in catalysis.
Some UCH enzymes have significant C-terminal extensions. The functions of the
C-terminal
extensions are still unknown but appear to be involved in proper localization
of the enzyme.
The active site of these UCH enzymes contains a catalytic triad consisting of
cysteine,
25 histidine, and aspartate and utilizes a chemical mechanism similar to that
of papain. The
crystal structure of one of these, UCH-L3, has been solved at 1.~ 1~
resolution. The enzyme
comprises a central antiparallel 13-sheet flanked on both sides by helices.
The 13-sheet and one
of the helices are similar to those observed in the thiol protease cathepsin
B. The similarity
includes the three amino acid residues that comprise the active site, Cys95,
Hisls9, and Aspl84.
3o The active site appears to fit the binding of ubiquitin that may anchor
also at an additional site.
The catalytic site in the free enzyme is masked by two different segments of
the molecule that
limit nonspecific hydrolysis and must undergo conformational rearrangement
after substrate
binding.
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UBP (type 2 UCH) enzymes are capable of cleaving the ubiquitin gene products
and
disassembling polyubiquitin chains after hydrolysis. It appears that there is
a core region of
about 450 amino acids delimited by CYS and HIS boxes. Many of these isoforms
have N-
terminal extensions and a few have C-terminal extensions. In addition, there
are variable
sequences in the core region of many of the isoforms. The functions of these
divergent
sequences remain poorly characterized. Another interesting function of
specific UBPs is the
regulation of cell proliferation. It was observed that cytokines induced in T-
cells specific de-
ubiquitinating enzymes (DUBS), termed DUB-1 and DUB-2. DUB-1 is induced by
stimulation
l0 of the cytokine receptors for IL-3, IL-5, and GM-CSF, suggesting a role in
its induction for the
J3-common (betac) subunit of the interleukin receptors. Overexpression of a
dominant negative
mutant of JAK2 inhibits cytokine induction of DUB-1, suggesting that the
regulation of the
enzyme is part of the cell response to the JAI~/STAT signal transduction
pathway. Continued
expression of DUB-1 arrests cells at Gl; therefore, the enzyme appears to
regulate cellular
15 growth via control of the Go-Gl transition. The catalytic conserved Cys
residue of the enzyme
is required for its activity. DUB-2 is induced by IL-2 as an immediate early
(IE) gene that is
down-regulated shortly after the initiation of stimulation. The function of
this enzyme is also
obscure. It may stimulate or inhibit the degradation of a critical cell-cycle
regulator.
20 Cytokines, such as interleukin-2 (IL-2), activate intracellular signaling
pathways via
rapid tyrosine phosphorylation of their receptors, resulting in the activation
of many genes
involved in cell growth and survival. The deubiquitinating enzyme DUB-2 is
induced in
response to IL-2 and is expressed in human T-cell lymphotropic virus-I (HTLV-
1)-
transformed T cells that exhibit constitutive activation of the IL-2 JAI~/STAT
(signal
25 transducers and activators of transcription) pathway, and when expressed in
BalF3 cells DUB-
2 markedly prolonged IL-2-induced STATE phosphorylation. Although DUB-2 does
not
enhance TL-2-mediatedproliferation, when withdrawn from growth factor, cells
expressing
DUB-2 had sustained STATE phosphorylation and enhanced expression of IL-2-
induced genes
cis and c-myc. DUB-2 expression markedly inhibited apoptosis induced by
cytokine
30 withdrawal allowing cells to survive. Therefore, DUB-2 has a role in
enhancing signaling
through the JAK/STAT pathway, prolonging lymphocyte survival, and, when
constitutively
expressed, may contribute to the activation of the JAK/STAT pathway observed
in some
transformed cells. (Migone, T.-S., et al., Blood. 2001;98:1935-1941).
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Protein ubiquitination is an important regulator of cytokine-activated signal
transduction pathways and hematopoietic cell growth. Protein ubiquitination is
controlled by
the coordinate action of ubiquitin-conjugating enzymes and deubiquitinating
enzymes.
Recently a novel family of genes encoding growth-regulatory deubiquitinating
enzymes
(DUB-1 and DUB-2) has been identified. DUBS are immediate-early genes and are
induced
rapidly and transiently in response to cytokine stimuli. By means of
polymerase chain reaction
amplification with degenerate primers for the DUB-2 complementary DNA, 3
marine bacterial
artificial chromosome (BAC) clones that contain DUB gene sequences were
isolated. One
BAC contained a novel D UB gene (D UB-2A) with extensive homology to D UB-2.
Like D UB-
1 and D UB-2, the D UB-2A gene consists of 2 exons. 'The predicted DUB-2A
protein is highly
related to other DUBS throughout the primary amino acid sequence, with a
hypervariable
region at its C-terminus. W vitro, D UB-2A had functional deubiquitinating
activity; mutation
of its conserved amino acid residues abolished this activity. The 5' flanking
sequence of the
DUB-2A gene has a hematopoietic-specific functional enhancer sequence. It is
proposed that
there are at least 3 members of the D UB subfamily (D UB-1, D UB-2, and D UB-
2A) and that
different hematopoietic cytokines induce specific DUB genes, thereby
initiating a cytokine-
specific growthresponse. (Baek , I~.-H., et al, Blood. 2001;9:636-642).
Protein ubiquitination also serves regulatory functions in the cell that do
not involve
proteasome-mediated degradation. For example, Hicke and Riezman have recently
demonstrated ligand-inducible ubiquitination of the Stet receptor in yeast.
Ubiquitination of
the Stet receptor triggers receptor endocytosis and receptor targeting to
vacuoles, not
proteasomes. Also, Chen et al. have demonstrated that activation of the IB
kinase requires a
rapid, inducible ubiquitination event. This ubiquitination event is a
prerequisite for the specific
phosphorylation of IB and does not result in subsequent proteolysis of the
kinase complex. The
ubiquitination of Stet and IB kinase appears reversible, perhaps resulting
from the action of a
specific deubiquitinating enzyme.
A large superfamily of genes encoding deubiquitinating enzymes, or UBPs, has
recently been identified. UBPs are ubiquitin-specific thiol-proteases that
cleave either linear
ubiquitin precursor proteins or post-translationally modified proteins
containing isopeptide
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ubiquitin conjugates. The large number of UBPs suggests that protein
ubiquitination, like
protein phosphorylation, is a highly reversible process that is regulated in
the cell.
Interestingly, UBPs vary greatly in length and structural complexity,
suggesting
functional diversity. While there is little amino acid sequence similarity
throughout their
coding region, sequence comparison reveals two conserved domains. The Cys
domain contains
a cysteine residue that serves as the active enzymatic nucleophile. The His
domain contains a
histidine residue that contributes to the enzyme's active site. More recent
evidence
demonstrates six homology domains contained by all members ofthe ubp
superfamily.
Mutagenesis of conserved residues in the Cys and His domains has identified
several residues
that are essential for UBP activity.
Recently, a growth regulatory deubiquitinating enzyme, DUB-1, that is rapidly
induced
in response to cytokine receptor stimulation was identified. DUB-1 is
specifically induced by
the receptors for IL-3, granulocyte macrophage-colony-stimulating factor, and
IL-5,
suggesting a specific role for the c subunit shared by these receptors. In the
process of cloning
theDUB-1 gene, a family of related, cross-hybridizing DUB genes was
identified. From this,
other DUB genes might be inducedby different growth factors. Using this
approach, an IL-2-
inducible DUB enzyme, DUB-2 and closely related DUB-2a were identified. DUB-1
and
2o DUB-2 are more related to each other than to other members of the ubp
superfamily and
thereby define a novel subfamily of deubiquitinating enzymes.
Hematopoietic-specific, cytokine induced DUBs in marine system have shown to
prolong cytokine receptor, see Migone, T. S., et al. (2001). The
deubiquitinating enzyme
DUB-2 prolongs cytokine-induced signal transducers and activators of
transcription activation
and suppresses apoptosis following cytokine withdrawal, Blood 9~, 1935-41;
Zhu, Y., et al.,
(1997). DUB-2 is a member of a novel family of cytokine-inducible
deubiquitinating
enzymes, J Biol Chem 272, 51-7 and Zhu, Y., et al., (1996). The marine DUB-1
gene is
specifically induced by the betac subunit of interleukin-3 receptor, Mol Cell
Biol 16, 480~-
17.). These effects are likely due to the deubiquitination of receptors or
other signaling
intermediates by DUB-1 or DUB-2, marine analogs of hDUBs. Inhibition of hDUBs
may
achieve downregulation of specific cytokine receptor signaling, thus
modulating specific
immune responses.
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Cytokines regulate cell growth by inducing the expression of specific target
genes. A
recently identified a cytokine-inducible, immediate-early gene, DUB-1, encodes
a
deubiquitinating enzyme with growth regulatory activity. In addition, a highly
related gene,
DUB-2, that is induced by interleukin-2 was identified. The DUB-2 mRNA was
induced in T
cells as an immediate-early gene and was rapidly down-regulated. Like DUB-1,
the DUB-2
protein had deubiquitinating activity iya vitf-o. When a conserved cysteine
residue of D UB-2,
required for ubiquitin-specific thiol protease activity, was mutated to serine
(C60S),
deubiquitinating activitywas abolished. DUB-1 and DUB-2 proteins are highly
related
throughout their primary amino acid sequence except for a hypervariable region
at their
COOH terminus. Moreover, the DUB genes co-localize to a region of mouse
chromosome 7,
suggesting that they arose by a tandem duplication of an ancestral DUB gene.
Additional DUB
genes co-localize to this region, suggesting a larger family ofcytokine-
inducible DUB
enzymes. We propose that different cytokines induce specific DUB genes. Each
induced DUB
l5 enzyme thereby regulates the degradation or the ubiquitination state of an
unknown growth
regulatory factor, resulting in a cytokine-specific growth response.
On the basis of these structural criteria, additional members of the DUB
subfamily can be
identified in the GenBank~. The highest degree of homology is in the Cys and
His domains.
Additionally, this putative humanDUB protein contains a Lys domain (amino
acids 400-410)
zo and ahypervariable region (amino acids 413-442).
Murine DUB (mDUB) subfamily members differ from other IJBPs by functional
criteria as well. mDUB subfamily members are cytokine-inducible, immediate-
early genes and
may therefore play regulatory roles in cellular growth or differentiation.
Also, DUB proteins
25 are unstable and are rapidly degraded by ubiquitin-mediated proteolysis
shortly after their
induction.
mDUB reports demonstrate that specific cytokines, such as IL-2 and IL-3,
induce
specific deubiquitinating enzymes (D UBs). The D UB proteins may modify the
ubiquitin-
3o proteolytic pathway and thereby mediate specific cell growth or
differentiation signals. These
modifications are temporally regulated. The DUB-2 protein, for instance, is
rapidly but
transiently induced by IL-2. Interference of D UB enzymes with specific
isopeptidase
inhibitors may block specific cytokine signaling events.
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The prior art teaches some partial sequences with homology to DUBS;
specifically
Human cDNA sequence SEQ m N0:17168 in EP1074617-A2; a human protease and
protease inhibitor PPIM-4 encoding cDNA; in W0200110903-A2 and human ubiquitin
protease 23431 coding sequence in W0200123589-A2.
References
1. Baek, K. H., Mondoux, M. A., faster, R., Fire-Levin, E., and D'Andrea, A.
D. (2001). DUB-2A, a
new member of the DUB subfamily of hematopoietic deubiquitinating enzymes,
Blood 98, 636-42.
LO 2. faster, R., Baek, K. H., and D'Andrea, A. D. (1999). Analysis of cis-
acting sequences and trans-
acting factors regulating the interleukin-3 response element of the DUB-1
gene, Biochim Biophys Acta
1446, 308-16.
3. faster, R., Zhu, Y., Pless, M., Bhattacharya, S., Mathey-Prevot, B., and
D'Andrea, A. D. (1997).
JAK2 is required for induction of the murine DUB-1 gene, Mol Cell Biol 17,
3364-72.
15 4. Migone, T. S., Humbert, M., Rascle, A., Sanden, D., D'Andrea, A.,
Johnston, J. A., Baek, K. H.,
Mondoux, M. A., faster, R., Fire-Levin, E., et al. (2001). The
deubiquitinating enzyme DUB-2
prolongs cytokine-induced signal transducers and activators of transcription
activation and suppresses
apoptosis following cytokine withdrawal, Blood 98, 1935-41.
Zhu, Y., Carroll, M., Papa, F. R., Hochstrasser, M., and D'Andrea, A. D.
(1996a). DUB-1, a
~0 deubiquitinating enzyme with growth-suppressing activity, Proc Natl Acad
Sci U S A 93, 3275-9.
6. Zhu, Y., Lambert, K., Corless, C., Copeland, N. G., Gilbert, D. J.,
Jenkins, N. A., and D'Andrea, A.
D. (1997). DUB-2 is a member of a novel family of cytokine-inducible
deubiquitinating enzymes, J
Biol Chem 272, 51-7.
7. Zhu, Y., Pless, M., Inhorn, R., Mathey-Prevot, B., and D'Andrea, A. D.
(1996b). The rnurine DUB-1
25 gene is specifically induced by the betac subunit of interleukin-3
receptor, Mol Cell Biol 16, 4808-17.
Scott Emr described a role for monoubiquitination in protein sorting in the
late endosome,
which has a role in determining which proteins, both newly synthesized and
endocytosed, will
be delivered to the lumen of the vacuole and which to its limiting membrane.
Proteins
3o destined for lumen are sorted into internal vesicles at the multivesicular
body (MVB) stage of
endosome maturation, whereas proteins destined for the vacuolar membrane, or
for recycling
to the plasma membrane, remain in the endosome's limiting membrane. Emr showed
that the
sorting of a vacuolar hydrolase into MVB vesicles requires the
monoubiqutination of this
cargo molecule at a specific lysine residue (Katzmann et al., 2001). Thus,
monoubiquitination
35 is a green light for traffic to proceed from this important intracellular
intersection to the lumen
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of the vacuole. The policeman directing the traffic is an endosome-localized
protein complex
called ESCRT-I, one of whose components, Vps23, plays a key role in
recognizing the cargo's
ubiquitin signal (I~atzmann et al., 2001). Vps23 is one of a small family of
UEV proteins
(ubiquitin E2 variants) that resemble E2s but cannot perform canonical E2
functions. The
ESCRT-I complex binds ubiquitin, and a mutation in Vps23 that cripples
ubiquitin-dependent
sorting in the MVB pathway abolishes ubiquitin binding to ESCRT-I. A model in
which
Vsp23 binds ubiquitin directly, while still inferential, received support from
structural studies
of a different UEV protein. Intriguingly, the mammalian homolog of Vps23,
known as tsg101,
is a tumor suppressor (Li and Cohen, 1996) The current results suggest that
mutations in
tsg101 could cause persistent signaling by growth factor receptors because of
inappropriate
receptor recycling to the plasma membrane, thus leading to tumorigenesis.
A role for monoubiquitination in triggering the first step of endocytosis-the
internalization of
plasma membrane proteins-is well established (Hicke, 2001), but how this
signal is
recognized has been unclear. Linda Hicke reported that yeast Entl is vital for
the ubiquitin-
dependent endocytosis of yeast factor receptor (see also Wendland et al.,
1999). Entl carries a
proposed ubiquitin binding motif called the UllVI domain (Hofinann and
Falquet, 2001 ), and
Hicke showed that Entl indeed binds ubiquitin directly. Entl also binds
clathrin (Wendland et
al., 1999) and so is poised to link monoubiquitinated cargo molecules to the
endocytic
machinery. Hicke's and Emr's results suggest that the ability of monoubiquitin
to signal two
different trafficking outcomes relies in part on distinct localizations of the
relevant signal-
recognizing components-Entl resides at the plasma membrane, while ESCRT-I is
associated
with late endosomes.
'S Fanconi Anemia (FA) is a rare cancer susceptibility disorder associated
with cellular
sensitivity to DNA damage that can be caused by mutations in at least seven
genes. Alan
D'Andrea shed new light on the molecular basis of FA: monoubiquitination of a
specific
lysine residue in one FA protein, known as D2, requires the activities of four
upstream FA
genes and leads to the relocalization of D2 within the nucleus (Garcia-Higuera
et al., 2001 ).
30 In normal cells, monoubiquitination of D2 is strongly augmented following
DNA damage and
is strictly required for damage-associated targeting of D2 and BRCAl to
subnuclear foci.
Thus, D2 monoubiquitination links an FA protein complex to the BRCAl repair
machinery.
Although the downstream events in this pathway are still unclear, localization
of the signal-
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11
recognizing factors) will likely be critical. This new function of ubiquitin
carnes a strong
flavor of certain roles of Sumo-1, a UbL that has been implicated in protein
targeting to
specific subnuclear structures (Hochstrasser, 2000).
Polyubiquitin chains are well known as a signal for substrate destruction by
26S proteasomes.
But there are several kinds of chains, linked through different lysines of
ubiquitin, suggesting
that different chains might be distinct signals (Pickart, 2000). James Chen
provided rigorous
proof of this hypothesis by showing that noncanonical polyubiuqitination can
activate
phosphorylation-in contrast to numerous examples of the converse regulation
(Hershko and
to Ciechanover, 1998). Postreplicative DNA repair and the activation of IkBa,
kinase (IKK)
require chains linked through Lys63, rather than the Lys48-chains that usually
signal
proteasomal proteolysis. Chen found that Takl kinase is a downstream target of
Lys63-chain
signaling in the II~K activation pathway. The assembly of these chains depends
on an unusual
UEV/E2 complex and a RING finger protein, Traf6 (Deng et al., 2000). (The RING
finger
defines a large E3 family.) Modification of Traf6 with a Lys63-chain leads to
the activation of
Takl, which in turn phosphorylates IKI~ (Wang et al., 2001). Activated IKKK
then
phosphorylates IkBa and triggers its tagging with Lys48-chains. Only then do
proteasomes
enter the picture-they degrade IkBa, and thereby free its partner, NFkB, to
translocate to the
nucleus and activate the expression of inflammatory response genes. Chen's
results suggest
z0 that Traf6 is the target of the Lys63-chain, as well as a catalyst of its
assembly. Indeed, many
other RING E3s also self modify-although the consequence is more apt to be
suicide (cf.
tagging with Lys48-chains) than the kind of personality change seen with Traf6
(Joazeiro and
Weissman, 2000). It remains to be seen if a similar mechanism applies in DNA
repair, where a
different RING protein, the RadS helicase, binds to a related UEV/E2 complex
(Ulrich and
Jentsch, 2000). New genetic data reported by Helle Ulrich confirmed the
central importance of
RadS in Lys-63 chain signaling in DNA repair (Ulrich, 2001).
These reports suggest a variety of new functions of protein ubiquitination and
its potential
involvement of subcellular trafficking including nucleus and the lumen of the
intracellular
3o vesicles. Thus regulation of ubiquitination by deubiquitinating proteases
in various subcellular
localization is become a critical issue.
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12
Recently, a number of proteins have been identified as capable of transducing,
that is, moving
across cellular and nuclear membranes in an energy-independent manner.
Transducing
sequences have been identified in proteins involved in circadian rhythm, such
as human
Period proteins. It is thought that these proteins move more freely through
cellular and
nuclear membranes, and that this movement permits concerted control. No other
enzymes
involved in the deubiquitination activities have been identified as being
capable of transducing
or having NLS until now.
The presence of an NLS at the C-terminal suggests that the hDUB7 and its
marine ortholog,
to mDUB7, are capable of translocating to the nucleus, possibly by importin-
dependent manner
and that these DUBS have a role in deubiquitinating ubiquitinated nuclear
proteins andlor
ubiquitinated proteins that are translocated to the nucleus. This has never
been identified
before. Protein ubiquitination targets selectively to proteasome degradation
and/or provides
facilitating protein localization. Thus, nuclear protein deubiquitination may
have a role in
l5 unique function in regulation of nuclearprotein degradation as well as
nuclear protein
localization. The same logic can be applied to the vesicular targeting of DUB7
by targeting
sequence, regulating vesicular protein degradation as well as invloved in
traficking of
vesicular proteins.
ao References
Katzmann D.J., Babst M. and Emr S.D. (2001) LTbiquitin-dependent sorting into
the
multivesicular body pathway requires the function of a conserved endosomal
protein sorting
complex, ESCRT-I. Cell, 106:145-155.
Li L. and Cohen S.N. (1996) tsg101: a novel tumor susceptibility gene isolated
by controlled
Z5 homozygous functional knockout of allelic loci in mammalian cells. Cell,
85:319-329.
Hicke L. (2001) A new ticket for entry into budding vesicles-ubiquitin. Cell,
106:527-530.
Wendland B., Steece K.E. and Emr S.D. (1999) Yeast epsins contain an essential
N-terminal
ENTH domain, bind clathrin, and are required for endocytosis. EMBO J., 18:4383-
4393.
Hofinann K. and Falquet L. (2001) A ubiquitin-interacting motif conserved in
components of
3o the proteasomal and lysosomal protein degradation systems. T~ehds Biochem.
Sci., 26:347-
350.
CA 02479647 2004-09-16
WO 03/083050 PCT/US03/08590
13
Garcia-Higuera L, Taniguchi T., Ganesan S., Meyn M.S., Timmers C., Hejna J.,
Grompe M.
and D'Andrea A.D. (2001) Interaction of the Fanconi Anemia proteins and BRCAl
in a
common pathway. Mol. Cell, 7:249-262.
Hochstrasser M. (2000) Evolution and function of ubiquitin-lilce protein-
conjugation systems.
Nat. Cell Biol., 2:E153-E157.
Pickart C.M. (2000) Ubiquitin in chains. Trehds Biochem. Sci., 25:544-548.
Pickart C.M. (2000) Ubiquitin in chains. Ti°ends Bioclzem. Sci.,
25:544-548.
Hershko A. and Ciechanover A. (1998) The ubiquitin system. Af~nu. Rev.
Biochem., 67:425-
479.
to Deng L., Wang C., Spencer E., Yang L., Braun A., You J., Slaughter C.,
Pickart C. and Chen
Z.J. (2000) Activation of the IkB kinase complex by TRAF6 requires a dimeric
ubiquitin-
conjugating enzyme complex and a unique polyubiquitin chain. Cell, 103:351-
361.
Wang C., Deng L., Hong M., Akkaraju G.R., moue J.-I. and Chen Z.J. (2001) TAKl
is a
ubiquitin-dependent kinase of MKK and IKK. Nature, 412:346-351.
Joazeiro C.A.P. and Weissman A.M. (2000) RING finger proteins: mediators of
ubiquitin
ligase activity. Cell, 102:549-552.
Ulrich H. (2001) The srs2 suppressor of UV sensitivity acts specifically on
the RADS- and
MMS2-dependent branch of the RAD6 pathway. Nucleic Acids Res., 29:3487-3494.
Ulrich H.D. and Jentsch S. (2000) Two RING finger proteins mediate cooperation
between
ubiquitin-conjugating enzymes in DNA repair. EMBO J., 19:3388-3397.
Summary of the Invention
The present invention is directed to identification of human homolog of marine
DUBS,
hematopoietic-specific, cytol~ine-inducible deubiquitinating proteases found
on chromosome
7, respective regulatory region and its marine ortholog, named as hDUB7 and
mDUB7,
respectively. Both hDUB7 and its marine ortholog mDUB7 were identified by
searching
human and mouse genome databases using marine DUB-1 and DUB-2 sequences. These
genes (hDUB7 and mDUB7) share open reading frames (ORFs) that are 67% amino
acid
identity to each other, when gaps caused by deletion was not counted as
mismatch, and
exhibit 75% identity in nucleotide sequence. Furthermore, both hDUB7 and mDUB7
share
48% identity to marine DUBs, DUB 1 and DUB2 within 297 amino acids core DUB
sequences. In addition, hDUB7 and mDUB7 genes share open reading frames that
are greater
than 92% amino acid identity within 540 amino acids N-terminal ubiquitin
protease domain
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(with 98.4% identity within 313 amino acid core). These genes also exhibit 74%
identity
within 138 amino acids C-terminal conserved domain containing several putative
nuclear
localization sequences (NLSs) and stretchs of amino acid sequences that is
known to possess
transducing capacity (h AKKHT~KSKKKI~I~SKDKHR and HRHKKKKKKKKRHSRK).
Therefore, the present invention is also directed to a transducing peptide
comprising an NLS
or transducing sequence of hDUB7 or mDUB7 linked to a cargo molecule. The
invention also
includes a transducing peptide comprsing an NLS or transducing sequence is
selected from the
group consisting of a peptidyl fragment comprising K AKKHKKSKKKKKSKDKHR,
HRHKKKKKKKKRHSRK, KKHKKSKKKI~KSI~DI~HR, and HRHRKKKKKKKR_H_SRI~.
to The invention also comprsises a transducing peptide wherein the cargo
molecule is a
biologically active protein, therapeutically effective compound, antisense
nucleotide, or test
compound. The invention also includes a method of delivering a biologically
active protein,
therapeutically effective compound, antisense nucleotide, or test compound to
a cell wherein a
transducing peptide is added exogenously to a cell.
Manipulation of these gene products by small molecular compounds can (1)
reduce
inflammation by regulating proinflammatory cytokine signaling, (2) modulate
autoimmune
diseases by regulating cytokine receptor signaling that are critical for
lymphocytes
proliferation, and (3) immure over-reaction during infection using above
mechanisms.
Search methods for identifyi~ hDUB7 and mDUB7:
mDUBl (LT41636), mDUB2 (U70368), and mDUB2A (AF393637) DNA sequences were
used to search against nr (All non-redundant GenBank CDS
translations+PDB+SwissProt+PIR+PRF) in GenBank for potential homologs.
Homology was
found to a cDNA (AI~022759) whose C terminal was incomplete (3660 nucleotides
capable of
expressing 1197 amino acids run-off translation). In order to in silico clone
the full length.
Both EST extending and genomic sequence annotation methods were used. Sequence
of
AK022759 was searched against human ESTs and genomic sequences. AK022759 was
extended manually based on matching ESTs and mapped genomic sequence on contig
NT 007844.8 from chromosome 7. From these full-length sequence for open
reading frame
for hDUB7 was generated (3951 nucleotides long DNA segment capable of
generating 1316
amino acids long polypeptide).
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For in silico cloning of the putative full length of mDUB7, hDUB7 amino acid
sequence was
used to search against nr by blastp. The highest match to Mouse proteins is a
protein similar
to mDUB2. The accession number for this protein is BAB27190 and for nucleotide
sequence
is AK010801 (1485 nucleotide long capable of translating 487 amino acids run-
on
translation). Based on Genbank annotation, the gene has partial sequence with
C terminal
incomplete. In order to get the full length of mDUB7, nucleotide sequence of
AK010801 was
used to search against Mouse Genomic sequence. There was no match to Mouse
curated NT
contigs _ -database and match was found on contig 70795 from Mouse Arachne
Nov30
database (preliminary assembly of the mouse WGS reads based on an Nov 9th
freeze of the
WGS -data) in Genbank. Putative genes from contig_70795 were annotated by
GENSCAN
prediction. There is one putative protein with extended/finished C terminal
aligned perfectly
with BAB27190 except having 33 amino acids missing in the middle of sequence.
The
nucleotide sequence of the 33 amino acids segment from BAB27190 was searched
against the
Mouse genomic sequence and found it matched to the genomic sequence region
that generates
the putative full length mDUB7 and has potential splice sites on the borders.
It implies that
exon was missed by GENSCAN annotation. A full length mouse DUB7 was
constructed by
adding 33 amino acids to the putative protein according to the genomic
sequence alignment
(3981 nucleotides long open reading frame capable of generating 1326 amino
acids long
polypeptide). The final mDUB7 sequence was aligned with hDUB7 and showed 67%
,o homology in amino acid level and 75% homology in nucleotide level.
TaqMan peal time PCR analysis of expYession of hDUB7 in human immunocytes upon
various stimulation
t5 Protocol of reverse transcription (RT) from total cellular RNA using random
hexamer as
primer (using TaqMan Reverse Transcription Reagents Cat# N808-0234)
1 ug of total RNA preparation in 100 ul of lxTaqMan RT Buffer Mix, 5.5mM MgCl2
, 0.5
mM dNTPs, 2.5 uM Random Hexamers, 40 U RNAse inhibitor, 125U Multiscribe
Reverse
30 Transcriptase. Mix by pipeting up and down. Incubate 25°C for 10
minutes (annealing step),
48°C for 30 minutes (reverse transcription), and 95°C for 5
minutes (heat killing of the
enzyme). The samples can be left at the machine at 4°C, or
alternatively, can be stored at -
20°C. Yield of cDNA synthesis can be measured by incorporation of small
portion of
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16
radioactive dATP (or dCTP). Average efficiency for this protocol is between 60-
80% of
conversion of RNA to cDNA.
Protocol of TaqMan real-time quantitative PCR
1 ul of TaqMan RT product in 12.5 ul of lx master Mix (Applied Biosystems Cat#
4304437)containing all necessary reaction components except primers and
probes, 0.9 uM
forward primer , 0.9 uM reverse primer, 0.2 uM probe. Mix by pipetting up and
down.
Samples containing GADPH primer pair and probe were also prepared as control.
Thermal
to cycling and detection of the real-time amplification were performed using
the ABI PRISM
7900HT Sequuence Detection System. The quantity of target gene is given
relative to the
GADPH control based on Ctvalues determined during the exponential phase of
PCR.
Primer-probe set used is as follow:
Forward Primer 5'-CCACGACAGAACTGCACTTGTAG-3'
Reverse Primer 5'- CCGGGACTTTCCATTTTCG-3'
Probe sequence 5'- CAACTGTAACCTCTCTGATCGGTTTCACGAA-3'
2o Table 1. Expression of hDUB7 in PBMC stimulated with LPS (100 ng/ml) for
1.5, land 24
hours by TaqMan (Donor 1).
LPS Stimulation/Time 1.5 hours 7 hours 24 hours
Fold Upregulation upon 0.9 ~ 1.5 1.0
stimulation
Table 2. Expression of DUB7 in PBMC stimulated with LPS (100 ng/ml) and/or PHA
(5
ug/ml) for 1.5, 7, 24 hour s by TaqMan (donor 2, donor 3)
Donor 2 Fold Upre
lation upon
stimulation
Stimuli/time LPS PHA LPS + PHA
1.5 hours 1.1 1.2 1.1
7 hours 3.3 9.2 9.2
24 hours 0.2 0.3 0.3
Donor 3 Fold U regulation
upon stimulation
Stimuli/time LPS PHA LPS + PHA
1.5 hours 1.2 1.0 1.2
7 hours 3.5 8.2 9.3
24 hours 0.5 0.5 0.6
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Table 3. Expression of hDUB7 in enriched B cells stimulated with LPS (100
ng/ml) or IL-4
and anti-CD40 mAb for 4 and 20 hours by TaqMan (Donor 4).
Donor 4 ~ Fold U rp egulation
upon stimulation
Stimuli/time LPS _ __IL-4, anti-CD40
mAb
4 hours 1.11 2.44
20 hours 0.70 1.0
Table 4. Expression of hDUB7 in entiched CD4+ T cells stimulated with anti-CD3
and anti-
CD28 mAbs for 3,6 and 18 hours by TaqMan (Donor 5).
mAbs Stimulation/Time 3 hours 6 hours 18 hours
Fold Upregulation upon 1.36 ~ 1.74 0.37
stimulation
to Table 5. Expression of hDUB7 in differentiated ThO, Thl and Th2 CD4+ T
cells (Day 4 after
differentiation) stimulated with anti-CD3 and anti-CD28 mAbs for 8 hours by
TaqMan
(Donor 6).
mAbs Stimulation ~ Th0 ~ Thl ~ Th2
Fold Upre~ulation upon stimulation 2.60 0.36 ~ 1.72
Table 6. Expression of hDUB7 in differentiated ThO, Thl and Th2 CD4+ T cells
(Day 7 after
differentiation) stimulated with anti-CD3 and anti-CD28 mAbs for 8 and 18
hours by TaqMan
(Donor 6).
mAbs Stimulation Th0 Thl Th2
Fold Upregulation in 8 hours1.38 1.11 1.71
Fold Upregulation in 18 0.94 0.81 1.47
hours
Table 7. Expression of hDUB7 in various tissue examined by Affymatrix chip
analysis
Tissue Relative
Intensity
Con Adipose_12287
Con Adipose 4190
2
CV_Heart_1 2545
CV_Heart_2 3907
CV_Heart 3 5367
CV Pericardia_13682
Dig Colon 2387
1
Dig Colon 2894
2
Dig Esophagus_15004
Dig Esophagus1658
2
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Dig FetalLiver_1 1288
Dig FetalLiver_2 4676
Dig FetalLiver_3 829
Dig FetalLiver_4 3161
Dig Liver 1 3094
Dig Liver 2 1527
Dig Liver 3 3410
Dig Pancreas_l 3731
Dig Pancreas 2 4837
Dig Rectum_1 2329
Dig Rectum 2 1851
Dig SalivaryGland_12337
Dig SalivaryGland_22110
Dig SmallIntestine_12838
Dig SmallIntestine_22662
Dig Stomach_1 2187
End_AdrenalGland_1591
End_AdrenalGland_22199
End Thyroid 1 2564
End Thyroid 2 2392
End Thyroid 3 3522
Exo_Breast_1 3673
Exo_Breast_2 6173
Exo_MammaryGland 3741
_1
Imm_BoneMarroW_1 1090
hnm Spleen 1 2429
hnm_Thymus_1 3666
Imm Thymus 2 1759
Rep Cervix_1 4482
Rep Cervix 2 3 3
62
Rep Placenta_1 1248
Rep Placenta_2 2378
Rep Placenta_3 1622
Rep Prostate_1 5128
Rep Prostate_2 2762
Rep Testis_l 2252
Rep Testis 2 3196
Rep Uterus_1 4720
Rep Uterus 2 3789
Res Lung 1 2313
Res Lung 2 3177
Res Lung 3 4409
Res Lung 4 2366
Res_Trachea_1 2152
Res_Trachea_2 2358
Res_Trachea_3 812
Res_Trachea_4 812
Sk_SkeletalMuscle_12838
Sk SkeletalMuscle6106
2
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Skin Skin 1 5500
Uri_Kidney 1 3593
Uri Kidney 2 1311
Uri Kidney 3 2747
Uri Kidney_4 1530
NS_Brain_1 3214
NS_Brain_2 2173
NS_Brain_3 1332
NS_Brain_4 2604
NS_Brain_5 1663
NS_Cerebellum_l3175
NS_Cerebellum_21766
NS_FetalBrain_14299
NS_FetalBrain_22549
NS_FetalBrain_34027
NS SpinalCord_l2976
NS SpinalCord_23999
NS SpinalCord 4614
3
Table 8. Expression of mDUB7 in various tissue examined by Affymatrix chip
analysis
Mouse Organ Relative Intentisty
#
A stomach 56
A stomach 11
B stomach 175
B stomach 97
C stomach 178
C stomach 126
A lymph 516
A lymph 365
B lymph 494
B lymph 335
C lymph 475
C lymph 509
A thymus 913
A thymus 1015
B thymus 881
B thymus 927
C thymus 834
C thymus 975
A prostate327
A prostate350
B prostate75
B prostate423
C prostate405
C prostate267
A uterus 549
A uterus 372
B uterus 225
B uterus 418
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C uterus 335
C uterus 401
Deubiquitination Assay
Confirmation that the DUB is a deubiquitinating enzyme may be shown using
5 previously identified deubiquitination assay of ubiquitin--galactosidase
fusion proteins, as
described previously in the literature. Briefly, a fragment of the DUB, of
approximately 1,500
nucleotides, based on the wild-type DUB cDNA (corresponding to amino acids 1
to about
500) and a cDNA containing a missense mutation are generated by PCR and
inserted, in
frame, into pGEX (Pharmacia), downstream of the glutathione S-transferase
(GST) coding
to element. Ub-Met--gal is expressed from a pACYC184-based plasmid. Plasmids
are co-
transformed as indicated into MC1061 Esche~ichia coli. Plasmid-bearing E. coli
MC1061
cells are lysed and analyzed by immunoblotting with a rabbit anti--gal
antiserum (Cappel), a
rabbit anti-GST antiserum (Santa Cruz), and the ECL system (Amersham Corp.).
in vitro
deubiquitinating enzyme activity may be shown from purified hDUB fusion
protein using
15 commercial polyubiquitinated protein as substrate.
HDUB7 and znDUB7 are potential inflanzatory cytokins specific Izzznzediate-
early genes
mDUB-I was originally cloned as an IL-3-inducible immediate-early gene.
Similarly,
mDUB-2 was cloned as an IL-2-inducible immediate-early gene. We examined
inducibility as
?o well as cell-type specific expression of these genes using Affymatrix-Chip
analysis and
multiple TaqMan analysis from human organ RNA samples and human immunocytes
RNA
samples. Our data suggest that expression of hDUB7 are not apparent in
monoocytes and
other myoloid cell types but high in fresh human PBMC from several donor.
Furthermore,
enriched cell populations of several lymphocytes, including B cells, CD4+ T
cells of Th-1 and
?5 Th-2 differentiation conditions as well as bulk CD4+ T cells showed
significant upregulation
upon appropriate stimulations. Currently, we can not rule out the possibility
of upregulation
upon stimulation in CD8+ T cells and potentially NI~/NK-T cells.
The DUB Subfamily of the ubp Superfamily
3o From these data we propose that hDUB4s and hDUB8s are members of a discrete
subfamily of deubiquitinating enzymes that shows the strongest similarity to
mDUB
subfamily including mDUBl, mDUB2, and mDUB2A, called the DUB subfamily. DUB
subfamily members contain distinct structural features that distinguish them
from other ubps.
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First, DUB subfamily members are comparatively small enzymes of approximately
500-550
amino acids. Second, D UB subfamily members share amino acid similarity not
only in the Cys
and His domains but also throughout theirprimary amino acid sequence. For
instance, DUB
proteins contain a lysine-rich region (Lys domain) and a HV domain near their
carboxyl
terminus.
The regulatory regions, or promoter regions, of each of the hDUB7 was analyzed
for
putative transcription factor binding motifs using TRANSFACFind, a dynamic
programming
method, see Heinemeyer, T., et al., "Expanding the TRANSFAC database towards
an expert
1o system of regulatory molecular mechanisms" Nucleic Acids Res. 27, 318-322,
(1999). The
Transfac database provides eukaryotic cis- and trans-acting regulatory
elements. The data is
shown as table X.
Table 9, putative transcription factor binding motifs within the hDUB7
regulatory or promoter
15 region. The position is indicated by nucleotides used in the table 9.
TransfacPosition(Score)Name Descri tion
M00148 1960..1966(100)SRY sex-determining region Y gene
product
876..870(100)
1357..1351
(92)
1881..1875(92)
1749..1755(90)
118..124(90)
267..261 (90)
275..269(90)
1663..1669(90)
1313..1319(90)
1860..1854(90)
108..114(90)
M00240 491..497(100) Nkx-2.5 homeo domain factor Nkx-2.5/Csx,
tinman
homolog
1512..1506(90)
1894..1888(90)
M00028 1844..1848(100)HSF heat shock factor (Drosophila)
1835..1839(100)
251..247(100)
265..261 (100)
273..269(100)
1429..1433(100)
1315..1319(100)
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22
1264..1268
100)
1060..1064
100)
1014..1010(100)
1540..1536(100)
1559..1555(100)
1619..1615(100)
110..114(100)
66..70(100)
1950..1946(100
1737..1741
95)
1635..1639(95)
651..647 95)
1103..1107(95
1082..1078(95
16..20(95)
1674..1678(94
1189..1185(94)
880..876(91)
M00029 247..243(100)
1667..1671
( 100)
1210..1206(100)
1745..1741
100)
71..75(100)
1844..1848(96)
1835..1839(96)
265..261 (96)
273..269(96)
1429..1433(96)
1315..1319(96)
1264..1268(96)
1060..1064(96)
1014..1010(96)
1540..1536(96)
1559..1555(96)
1619..1615(96)
110..114(96)
1950..1946(96)
1674..1678(95)
1189..1185(95)
1737..1741(93)
1635..1639(93)
651..647(93)
1103..1107(93)
1082..1078(93)
16..20(93)
1120..1124(90)
139..143(90)
M00101 1418..1412(
100)
1689..1695(98)
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23
1566..1572(98)
1460..1466(98)
1319..1325
98)
969..975(98)
1463..1457(98)
1614..1608(98)
1065..1059(94)
1599..1605(93)
1375..1369(93)
1840..1834(93)
1859..1865(92)
1168..1174(92)
1218..1212(92)
1478..1484(90)
M00048 447..452(100) ADRl alcohol dehydrogenase gene
regulator 1
535..540(95)
1716..1721(93)
459..454(93)
558..553(93)
1180..1185(93)
305..310(93)
38..43(92)
M00354 1951..1941 Dof3 Dof3 - single zinc finger transcription
99 factor
1560..1550(95)
104..114(93)
65..75(91)
M00227 1920..1928(98)v-Myb v-Myb
M00141 521..513(98) Lyf 1 LyF-1
828..820(98)
M00344 806..795(98) RAV1 3'-part ofbipartite RAV1 binding
site,
interacting with A.P2 domain
806..817(92)
1949..1960(92)
M00253 1139..1146(98)cap cap signal for transcription
initiation
681..688 96)
374..381 (96)
299..306(95)
1674..1667(94)
1737..1730(91)
31..24(91)
16..9(91)
1701..1694(91)
1909..1902(90)
619..626(90
1368..1375(90)
M00286 577..564(97) GKLF gut-enriched I~rueppel-like
factor
271..258(96)
M00199 684..676(96) AP-1 AP-1 binding site
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24
676..684(95)
M00183 227..218(96 c-Myb c-Myb
28..37(95
1247..1238(90)
M00154 1714..1721(96)STRE stress-response element
M00140 1824..1831(96 Bcd Bicoid
834..841 (93)
527..534(93)
M00100 1418..1412(96)CdxA CdxA
1209..1215
92)
1348..1354(91)
M00291 1652..1667(95)Freac-3 Fork head RElated Activator-3
M00073 1948..1958(95)deltaEFl deltaEF1
807..797(95)
1452..1442(92)
805..815(90)
M00216 1176..1167(95)TATA Retroviral TATA box
M00120 1952..1942(95)dl dorsal
1561..1551(93)
M00042 1861..1852(95 Sox-5 Sox-5
1790..1781(91)
M00174 675..685(95) AP-1 activator protein 1
M00230 1797..1808(95)Skn-1 maternal gene product
M00272 1024..1033(94)p53 tumor suppressor p53
103 3 ..1024(94)
M00160 1862..1851(94)SRY sex-determining region Y gene
product
M00022 111..120(94) Hb Hunchback
436..427(91)
584..575(91)
M00053 447..456(94) c-Rel c-Rel
M00249 1244..1256(93)CHOP- heterodimers of CHOP and C/EBPalpha
C/EBPalpha
M00142 1367..1362(93)NIT2 activator of nitrogen-regulated
genes
1348..1343(91)
M00289 1670..1658(93)HFH-3 HNF-3/Fkh Homolog 3 (= Freac-6)
M00019 1381..1366(93)Dfd Deformed
1593..1608(91)
M00147 1903..1912(92)HSF2 heat shock factor 2
M00184 806..815(92) MyoD myoblast determining factor
M00345 225..218(92) GAmyb GA-regulated myb gene from
barley
M00094 1658..1670(92)BR-C Broad-Complex Z4
1398..1386(90)
M00349 1200..1191 GATA-2 GATA-binding factor 2
(92)
M00077 443..451 (92) GATA-3 GATA-binding factor 3
M00087 388..399(91) Ik-2 Ikaros 2
M00099 1268..1283(91)S8 S8
M00285 1399..1411(91)TCF11 TCF11/KCR-F1/Nrfl homodimers
M00241 1224..1217(91)Nl~-2.5 homeo domain factor Nkx-2.5/Csx,
tinman
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homolog
1526..1519(91)
M00283 1863..1878(90)Zeste Zeste transvection gene product
M00046 1113..1105(90)GCRl GCRl
M00353 1069..1079 Dof2 DofZ - single zinc finger transcription
90) factor
1951..1941
(90)
M00263 985..994(90) StuAp Aspergillus Stunted protein
M00051 448..457(90) NF-kappaB NF-kappaB (p50)
M00350 1200..1191(90)DATA-3 GATA-binding factor 3
M00276 1851..1860(90)Matl-Mc M-box interacting with Matt-Mc
M00075 1936..1945(90)DATA-1 GATA-binding factor 1
442..451 (90)
M00355 279..269(90) PBF PBF (MPBF)
1897..1887(90)
M00352 1775..1785(90)Dofl Dofl / MNBla - single zinc finger
transcription
factor
M00294 1670..1658(90)HFH-8 HNF-3lFkh Homolog-8
M00131 1762..1748(90)HNF-3beta Hepatocyte Nuclear Factor 3beta
M00137 1320..1332(90)Oct-1 octamer factor 1
M00054 448 457(90) NF-kappaB NF-kappaB
~
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Table 10. Nucleotide sequence of coding region of human DUB7 (hDUB7)
ATGACCATAGTTGACAAAGCTTCTGAATCTTCAGACCCATCAGCCTATCAGAATC
AGCCTGGCAGCTCCGAGGCAGTCTCACCTGGAGACATGGATGCAGGTTCTGCCAG
CTGGGGTGCTGTGTCTTCATTGAATGATGTGTCAAATCACACACTTTCTTTAGGAC
CAGTACCTGGTGCTGTAGTTTATTCGAGTTCATCTGTACCTGATAAATCAA.A.ACCA
TCACCACAA.AAGGATCAAGCCCTAGGTGATGGCATCGCTCCTCCACAGAAAGTTC
TTTTCCCATCTGAGAAGATTTGTCTTAAGTGGCAACAAACTCATAGAGTTGGAGCT
GGGCTCCAGAATTTGGGCAATACCTGTTTTGCCAATGCAGCACTGCAGTGTTTAA
~o CCTACACACCACCTCTTGCCAATTACATGCTATCACATGAACACTCCAAA.ACATGT
CATGCAGAAGGCTTTTGTATGATGTGTACAATGCAAGCACATATTACCCAGGCAC
TCAGTAATCCTGGGGACGTTATTAAACCAATGTTTGTCATCAATGAGATGCGGCG
TATAGCTAGGCACTTCCGTTTTGGAAACCAAGAAGATGCCCATGAATTCCTTCAA
TACACTGTTGATGCTATGCAGAAAGCATGCTTGAATGGCAGCAATAAATTAGACA
is GACACACCCAGGCCACCACTCTTGTTTGTCAGATATTTGGAGGATACCTAAGATC
TAGAGTCAAATGTTTAAATTGCAAGGGCGTTTCAGATACTTTTGATCCATATCTTG
ATATAACATTGGAGATAAAGGCTGCTCAGAGTGTCAACAAGGCATTGGAGCAGTT
TGTGAAGCCGGAACAGCTTGATGGAGAAA.ACTCGTACAAGTGCAGCAAGTGTAA
AAAGATGGTTCCAGCTTCAAAGAGGTTCACTATCCATAGATCCTCTAATGTTCTTA
?o CACTTTCTCTGAAACGTTTTGCAAATTTTACCGGTGGAAAAATTGCTAAGGATGTG
AAATACCCTGAGTATCTTGATATTCGGCCATATATGTCTCAACCCAACGGAGAGC
CAATTGTCTACGTCTTGTATGCAGTGCTGGTCCACACTGGTTTTAATTGCCATGCT
GGCCATTACTTCTGCTACATAAAAGCTAGCAATGGCCTCTGGTATCAA.ATGAATG
ACTCCATTGTATCTACCAGTGATATTAGATCGGTACTCAGCCAACAAGCCTATGTG
?s CTCTTTTATATCAGGTCCCATGATGTGAAAAATGGAGGTGAACTTACTCATCCCAC
CCATAGCCCCGGCCAGTCCTCTCCCCGCCCCGTCATCAGTCAGCGGGTTGTCACCA
ACAAACAGGCTGCGCCAGGCTTTATCGGACCACAGCTTCCCTCTCACATGATAAA
GAATCCACCTCACTTAAATGGGACTGGACCATTGAAAGACACGCCAAGCAGTTCC
ATGTCGAGTCCTAACGGGAATTCCAGTGTCAACAGGGCTAGTCCTGTTAATGCTT
3o CAGCTTCTGTCCAAAACTGGTCAGTTAATAGGTCCTCAGTGATCCCAGAACATCCT
AAGAAACAAAAAATTACAATCAGTATTCACAACAAGTTGCCTGTTCGCCAGTGTC
AGTCTCAACCTAACCTTCATAGTAATTCTTTGGAGAACCCTACCAAGCCCGTTCCC
TCTTCTACCATTACCAATTCTGCAGTACAGTCTACCTCGAACGCATCTACGATGTC
AGTTTCTAGTAAAGTAACAAA.ACCGATCCCCCGCAGTGAATCCTGCTCCCAGCCC
3s GTGATGAATGGCAAATCCAAGCTGAACTCCAGCGTGCTGGTGCCCTATGGCGCCG
AGTCCTCTGAGGACTCTGACGAGGAGTCAAAGGGGCTGGGCAAGGAGAATGGGA
TTGGTACGATTGTGAGCTCCCACTCTCCCGGCCAAGATGCCGAAGATGAGGAGGC
CACTCCGCACGAGCTTCAAGAACCCATGACCCTAAACGGTGCTAATAGTGCAGAC
AGCGACAGTGACCCGAAAGAAAACGGCCTAGCGCCTGATGGTGCCAGCTGCCAA
4o GGCCAGCCTGCCCTGCACTCAGAA.AATCCCTTTGCTAAGGCAAACGGTCTTCCTG
GAAAGTTGATGCCTGCTCCTTTGCTGTCTCTCCCAGAAGACAAA.ATCTTAGAGAC
CTTCAGGCTTAGCAACAAACTGAAAGGCTCGACGGATGAAATGAGTGCACCTGG
AGCAGAGAGGGGCCCTCCCGAGGACCGCGACGCCGAGCCTCAGCCTGGCAGCCC
CGCCGCCGAATCCCTGGAGGAGCCAGATGCGGCCGCCGGCCTCAGCAGCACCAA
4s GAAGGCTCCGCCGCCCCGCGATCCCGGCACCCCCGCTACCAAAGAAGGCGCCTGG
GAGGCCATGGCCGTCGCCCCCGAGGAGCCTCCGCCCAGCGCCGGCGAGGACATC
GTGGGGGACACAGCACCCCCTGACCTGTGTGATCCCGGGAGCTTAACAGGCGATG
CGAGCCCGTTGTCCCAGGACGCAAAGGGGATGATCGCGGAGGGCCCGCGGGACT
CGGCGTTGGCGGAAGCCCCGGAAGGGTTGAGTCCGGCTCCGCCTGCGCGGTCGGA
so GGAGCCCTGCGAGCAGCCACTCCTTGTTCACCCCAGCGGGGACCACGCCCGGGAC
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GCTCAGGACCCATCCCAGAGCTTGGGCGCACCCGAGGCCGCAGAGCGGCCGCCA
GCTCCTGTGCTGGACATGGCCCCGGCCGGTCACCCGGAAGGGGACGCTGAGCCTA
GCCCCGGCGAGAGGGTCGAGGACGCCGCGGCGCCGAAAGCCCCAGGCCCTTCCC
CAGCGAAGGAGAA.AATCGGCAGCCTCAGAAAGGTGGACCGAGGCCACTACCGCA
GCCGGAGAGAGCGCTCGTCCAGCGGGGAGCCCGCCAGAGAGAGCAGGAGCAAG
ACTGAGGGCCACCGTCACCGGCGGCGCCGCACCTGCCCCCGGGAGCGCGACCGC
CAGGACCGCCACGCCCCGGAGCACCACCCCGGCCACGGCGACAGGCTCAGCCCT
GGCGAGCGCCGCTCTCTGGGCAGGTGCAGTCACCACCACTCCCGACACCGGAGCG
GGGTGGAGCTGGACTGGGTCAGACACCACTACACCGAGGGCGAGCGTGGCTGGG
o GCCGGGAGAAGTTCTACCCCGACAGGCCGCGCTGGGACAGGTGCCGGTACTACC
ATGACAGGTACGCCCTGTACGCTGCCCGGGACTGGAAGCCCTTCCACGGCGGCCG
CGAGCACGAGCGGGCCGGGCTGCACGAGCGGCCGCACAAGGACCACAACCGGGG
CCGTAGGGGCTGCGAGCCGGCCCGGGAGAGGGAGCGGCACCGCCCCAGCAGCCC
CCGCGCAGGCGCGCCCCACGCCCTCGCCCCGCACCCCGACCGCTTCTCCCACGAC
~5 "AGAACTGCACTTGTAGCCGGAGACAACTGTAACCTCTCTGATCGGTTTCACGAAC
ACGAA.A.ATGGAAAGTCCCGGAAACGGAGACACGACAGTGTGGAGAACAGTGACA
GTCATGTTGAAAAGAAAGCCCGGAGGAGCGAACAGAAGGATCCTCTAGAAGAGC
CTAAAGCAAAGAAGCACAAAAAATCAAAGAAGAAAAAGAAATCCAAAGACAAA
CACCGAGACCGCGACTCCAGGCATCAGCAGGACTCAGACCTCTCAGCAGCGTGCT
?o CTGACGCTGACCTCCACAGACAC~,AAAAAAAGAAGAAGAAAAAGAAGAGACATT
CAAGAAAATCAGAGGACTTTGTTAAAGATTCAGAACTGCACTTACCCAGGGTCAC
CAGCTTGGAGACTGTCGCCCAGTTCCGGAGAGCCCAGGGTGGCTTTCCTCTCTCTG
GTGGCCCGCCTCTGGAAGGCGTCGGACCTTTCCGTGAGAAAACGAAACACTTACG
GATGGAAAGCAGGGATGACAGGTGTCGTCTCTTTGAGTATGGCCAGGGTGATTGA
?5
Table 11. Deduced amino acid sequence of coding region of hDUB7
C-terminal potential nuclear localization (as well as targeting) sequences are
underlined.
MTIVDKASESSDPSAYQNQPGSSEAVSPGDMDAGSASWGAVSSLNDVSNHTLSLGPV
PGAVVYSSSSVPDKSKPSPQKDQALGDGIAPPQKVLFPSEKICLKWQQTHRVGAGLQ
NLGNTCFANAALQCLTYTPPLANYMLSHEHSKTCHAEGFCMMCTMQAHITQALSNP
GDVIKPMFVINEMRRIARHFRFGNQEDAHEFLQYTVDAMQKACLNGSNKLDRHTQA
TTLVCQIFGGYLRSRVKCLNCKGVSDTFDPYLDITLEIKAAQSVNKALEQFVKPEQLD
GENSYKCSKCKKMVPASKRFTIHRSSNVLTLSLKRFANFTGGKIAKDVKYPEYLDIRP
YMSQPNGEPIVYVLYAVLVHTGFNCHAGHYFCYIKASNGLWYQMNDSIVSTSD1RSV
LSQQAYVLFYIRSHDVKNGGELTHPTHSPGQSSPRPVISQRVVTNKQAAPGFIGPQLPS
4o HMII~NPPHLNGTGPLKDTPSSSMSSPNGNSSVNR.ASPVNASASVQNWSVNRSSVIPEH
PKKQKITISIHNKLLPVRQCQSQPNLHSNSLENPTKPVPSSTITNSAVQSTSNASTMSVSS
KVTKPIPRSESCSQPVMNGKSKLNSSVLVPYGAESSEDSDEESKGLGKENGIGTIVSSH
SPGQDAEDEEATPHELQEPMTLNGANSADSDSDPKENGLAPDGASCQGQPALHSENP
FAKANGLPGKLMPAPLLSLPEDKILETFRLSNKLKGSTDEMSAPGAERGPPEDRDAEP
4.5 QPGSPAAESLEEPDAAAGLSSTKKAPPPRDPGTPATKEGAWEAMAVAPEEPPPSAGE
DIVGDTAPPDLCDPGSLTGDASPLSQDAKGMIAEGPRDSALAEAPEGLSPAPPARSEEP
CEQPLLVHPSGDHARDAQDPSQSLGAPEAAERPPAPVLDMAPAGHPEGDAEPSPGER
VEDAAAPKAPGPSPAKEKIGSLRKVDRGHYRSRRERSSSGEPARESRSKTEGHI~HRRR
RTCPRERDRQDRHAPEHHPGHGDRLSPGERRSLGRCSHHHSRHRSGVELDWVRHHY
5o TEGERGWGREKFYPDRPRWDRCRYYFIDRYALYA.ARDWKPFHGGREHERAGLHERP
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HKDHNRGRRGCEPARERERHRPSSPRAGAPHALAPHPDRFSHDRTALVAGDNCNLSD
RFHEHENGKSRKRRHDSVENSDSHVEKKARRSEQKDPLEEPK AKKHi~KSKKKKKSK
DKHRDRDSRHQQDSDLSAACSDADLHRHKKKKKKKKRHSRKSEDFVKDSELHLPRV
TSLETVAQFRRAQGGFPLSGGPPLEGVGPFREKTKHLRMESRDDRCRLFEYGQGD
Table 12. Putative promoter sequence of hDUB7 (2 Kb sequence upstream of
initiation AUG)
GTAAAGTCTAAACTGAGAAGTGGAAGTGTGAACTGGCTGGAGGTGGAAGGTTGG
A.AAAGAGTCGGAGAA.AAGAACAGCATGTGCAGAGCCCAGAGACAGCAGGGACA
to AAAG CAAGACTTCAGCATGGTGGGAACGTGACGGAGAGGGTGTTT
GGCGAGGTTATTAGGTCAGACAATGTGAAGTCCAGACATTAAGATGTTGTGCTGT
GGGCAGTTGGGCCACTCCTGAAAGGTGTTCTTTCTTCCTTTCCTTTTCTTTCTTTCT
TTTCTTGAGGCAGAGTCTCTCTATGTCAGTCTGGAGTGCAGTGGCATGATCTCGGC
TCACTGCAATCTCTGCCTTCCAGGTTCAAGCAATTTTCCTTGCCTCAGCCTCCCAA
t5 GTAGCTGGGAATACAGGCGTGCGCCACCATGCCTGGTTAATTTTTTTATTTTTAGT
AGAGATGGGGTTTCCCCATGTTGGCCAGGCTGGTCTCGAACTCCTGGACTCAAGT
GATCCACCCACTTTGGCCTCCCAAAGTGCTGGGATTACAGGGGTGTGAGCCACTG
CGCCCCGCCCGGCCTTTTTTTTTTTTTTTTTTGAGACTTAATCTTGCTCTGTCACCA
AGGCTGGATATCAGTGGCACGGTTTTGGCTCTCTGCAACTTCTGTCTCCCAGGTTC
'o AAGCGATTTTCCTGACTCAGCCTCCCAAGTAGTTGAGATTACAGGTACGTGCCAC
CACGCCCGGCTAATTTTTGTATTTTTAGTAGAGATGAGGTTTCACTATGTTGGCCA
GACTGGTCTCAAACGCCTGACCTCAGGTGATTCACCTGCCTCGGCCTCCCAAAAT
GCTGGGATTACAGGTGTGCACCACCATGCCTGGGTAATTTTTGTTTTTCGTAGAGA
CAGGGTCTCACCATGTTGGCCAGGCTGGTCTCAAACTCCTGACCTCAAGCGATCT
?s GCCCACCTTGGCCTCCCAAGGTGCTGCAATTATAGGCATGAGCCACCGCGCCCGG
CCTCCTGAAAGGTTTTCTACATAGGAGTGGCATGTCTAGATGTGGCTACTGTTGGG
CGATTTTAGAAATATCCCTA.A.AAGCCTTCTGTTGACAGGGTGGCATAACCAGAAG
GAAGCCTGGCTGGGAACGCTGGACCTGGCTCTCAGTCCCAGTTGCTGACTGGTTG
CTTCATTTTATAGGCCCTGGGGATTCTGTCTGATCTCTCATACGTTCTTTATAAAA
3o ATTAAGTTAATGTATGTCCAGCAGTTGATGCAATGCCCAGTACATAGAA.AATGCT
CAATTAGTGGTAGCCCTAATATTTTAAAATAGGACTCAGAAAGAAAATTATAATC
AAGTCCTTTCATAACAGATATTTGTGTTTGAGTTTGATATCAGTAATGGCTTACGG
GTTTTATTTAAAAAGTCATACATTCCATATAAATGAGCCTCTTCAGAAAAATGGTT
TTAAAGGTGAGATCTCTATAATTATAATTTTA,AAA.A ATATAATGTATTTCACTTGG
35 TGCCATTTGCACTTTAAGCACAAA.ATTAAGTCTAGATTTTTTCTGTGTAGTTGATG
CTTTTCTCTGAGGAATTATACTCAAATTGAAGATGTAGTCAAATGTATTACTGTGT
ATAATTTTTCTAGTTTTAAGCAGTATAGAAGGAAAATATAGGTACTTAGTAAATA
AACAGAACTGAGAATTGAAATGTCCAATTATAAACTGAAATGCCAGACTTTTAGG
GGGCATGAAATGAAAATGAGAAGTTCTTTTAATCAAATACTTCACTGAAGATTTT
4o AAA.ATAAAGATTGTTGACATTCAGATTATCATGATGCTAAATGTCCCAAGGGGAT
TATTACAGAAATGTTAGAAAGTACTATTGTTTTTATATTTGAGTGATGTGTTTGAA
AATCACTTTAAA.ATGGCTGGAATGATCTTCCAAGATCTAACGGTAGGGTAAGGAG
ATTGCTTTTCTCACCTGATGAAACAAATACATACTTTTCATCTTTTGCAGAGTTGA
ACAATG
Table 13. Nucleotide sequence of coding region of marine DUB7 (mDUB7)
ATGACCATAGTTGACAAAACTGA.ACCTTCAGACCCATCAACCTGTCAGAACCAGC
so CTGGCAGTTGTGAGGCGGTCTCACCTGAAGACATGGACACAGGCTCTGCCAGCTG
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GGGCGCTGTGTCTTCAATAAGTGATGTCTCAAGTCACACACTTCCATTAGGGCCA
GTGCCTGGTGCTGTAGTTTATTCTAACTCGTCTGTACCTGAA.AAATCAAAGCCATC
ACCACCAAAGGATCAAGTCCTAGGTGATGGCATTGCTCCTCCTCAA.AAGGTCCTG
TTTCCATCTGAAAAGATTTGTCTTAAGTGGCAACAAAGTCATCGAGTTGGCGCTG
GGCTCCAGAATTTGGGCAACACCTGTTTTGCCAATGCCGCATTGCAGTGTCTGACT
TACACGCCACCCCTCGCCAATTACATGTTATCCCATGAACACTCCAAGACATGCC
ACGCAGAAGGATTTTGTATGATGTGCACGATGCAGACACACATTACCCAGGCACT
TAGCAACCCTGGGGATGTTATCAAGCCGATGTTCGTCATCAATGAAATGCGGCGT
ATAGCTAGACACTTCCGTTTTGGAAACCAAGAAGATGCCCATGAATTTCTTCAGT
io ACACGGTCGATGCCATGCAGAAAGCATGTTTAAATGGCAGCAATAAATTAGACA
GACACACCCAGGCCACCACCCTGGTCTGCCAGATATTTGGAGGCTACCTAAGATC
CCGAGTTAAATGTTTAAATTGCAAGGGTGTTTCAGATACCTTTGATCCATATCTGG
ACATAACGTTGGAGATTAAGGCTGCACAGAGTGTTACCAAGGCGTTAGAGCAGTT
TGTGAAGCCAGAACAACTGGATGGAGAAAACTCCTACAAGTGCAGCAAGTGCAA
~5 AAAAATGGTTCCAGCTTCAAAGAGATTCACAATCCATAGGTCCTCTAATGTTCTTA
CCATCTCACTGAAGCGCTTTGCCAACTTCACCGGTGGAAAGATTGCTAAGGATGT
GAAATATCCTGAGTACCTTGATATCCGGCCCTATATGTCTCAGCCCAATGGAGAG
CCAATTATTTATGTTTTGTATGCTGTGCTGGTGCACACTGGTTTTAATTGTCATGCT
GGCCACTACTTTTGCTACATCAAGGCTAGCAATGGCCTCTGGTATCAGATGAATG
~o ACTCCATCGTGTCCACCAGTGATATCAGAGCAGTGCTTAACCAGCAAGCTTACGT
GCTCTTTTATATCAGGTCCCATGATGTGAAA.AATGGAGGGGAGTCTGCTCATCCT
GCCCATAGCCCCGGCCAATCCTCTCCCCGCCCAGGAGTCAGTCAGCGGGTAGTCA
ACAACAAGCAGGTGGCTCCAGGGTTTATTGGACCCCAGCTGCCTTCCCATGTGAT
GAAGAACACGCCACACTTGAATGGCACCACGCCAGTGAAAGACACACCAAGTAG
as TTCTGTGTCAAGCCCTAACGGAAACACCAGCGTCAATAGGGCCAGTCCTGCTACT
GCTTCGACTTCTGTGCAGAACTGGTCTGTTACCAGACCCTCAGTTATTCCAGATCA
CCCCAAGAAACAAAAAATCACCATCAGTATTCACAACAAGTTGCCTGCTCGCCAG
GGTCAGGCACCACTGAATAACAGCCTCCATGGCCCTTGTCTGGAGGCTCCTAGTA
AGGCGGCACCCTCCTCCACCATCACTAACCCTTCTGCAATACAGTCTACCTCGAAC
3o GTACCCACAACGTCGACTTCCCCCAGTGAGGCCTGTCCCAAGCCCATGGTGAACG
GCAAGGCTAAAGTGGGCGCCAGTGTGCTTGTCCCCTATGGGGCCGAGTCCTCAGA
AGAGTCTGATGAGGAGTCGAAGGGCCTGGCCAAGGAGAACGGTGTGGACATGAT
GGCCGGCACTCACTCCGATAGGCCAGAAGCTGCTGCAGATGACGGTGCTGAGGCT
TCCTCCCATGAGCTTCAAGAACCCGTCCTGCTAAATGGTGCTAATAGCGCAGACA
~s GTGACTCACAAGAGAACAGCCTGGCATTTGACAGTGCCAGCTGCCAGGTCCAGCC
CGAGCTACACACAGAA.AACCTCTTTTCCAAACTTAATGGTCTTCCTGGAAAGGTG
ACGCCTGCTCCTTTGCAGTCTGTTCCTGAAGACAGAATCCTTGAGACCTTCAAGCT
TACCAACCAGGCAAAGGGTCCAGCGGGTGAAGAGAGTTGGACTACGACAGGGGG
AAGCTCTCCAAAGGACCCTGTTTCACAGCTGGAGCCCATCAGTGATGAGCCCAGT
a.o CCCCTTGAGATACCGGAGGCTGTCACCAATGGGAGCACACAGACCCCTTCCACCA
CATCACCCCTGGAGCCCACCATCAGCTGTACCAAAGAAGACTCGTCCGTTGTTGT
CTCAGCTGAACCTGTGGAGGGTTTGCCTTCCGTCCCTGCTCTTTGTAACAGCACTG
GTACTATCTTGGGGGATACCCCAGTGCCCGAATTGTGTGACCCTGGAGACTTGAC
TGCCAACCCGAGCCAGCCAACCGAAGCAGTGAAAGGTGATACAGCTGAGAAGGC
a.s TCAGGACTCTGCCATGGCTGAAGTGGTGGAGAGGCTGAGCCCTGCTCCCTCAGTA
CTCACAGGTGACGGGTGTGAGCAGAAACTCTTACTTTACCTCAGCGCAGAGGGGT
CAGAGGAGACAGAAGACTCTTCCAGAAGCTCGGCGGTCTCTGCTGACACGATGCC
CCCTAAGCCTGACAGGACCACCACCAGCTCCTGTGAAGGGGCTGCCGAGCAGGCT
GCTGGGGACAGAGGCGATGGAGGCCATGTGGGACCCAAAGCTCAGGAGCCTTCC
5o CCAGCCAAGGAAAAGATGAGCAGCCTCCGGAAAGTGGACCGAGGACACTATCGG
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AGCCGGAGAGAGCGCTCCTCCAGTGGGGAGCACGTGAGGGACAGCAGGCCCCGG
CCGGAGGACCATCACCATAAGAAGCGGCACTGCTACAGCCGAGAGCGGCCCAAG
CAGGACCGACACCCTACTAATTCATACTGCAATGGGGGCCAGCACTTGGGCCACG
GGGACAGAGCCAGCCCTGAGCGCCGCTCCCTGAGCAGGTATAGTCACCACCACTC
5 ACGGATTAGGAGTGGCCTGGAGCAGGACTGGAGCCGGTACCACCATTTGGAAAA
TGAGCATGCTTGGGTCAGGGAGAGATTCTACCAGGACAAGCTGCGGTGGGACAA
GTGCAGGTATTACCACGACAGGTACACGCCCCTATACACGGCCCGGGACGCCCGA
GAATGGCGGCCTCTGCATGGTCGTGAGCATGACCGCCTTGTCCAGTCTGGACGGC
CATACAAGGACAGCTACTGGGGCCGCAAGGGCTGGGAGCTGCAATCCCGGGGGA
o AGGAACGGCCCCACTTCAACAGCCCCCGAGAGGCCCCTAGCCTTGCTGTGCCCCT
CGAGAGACATCTCCAAGAGAAGGCTGCGCTGAGTGTGCAGGACAGCAGCCACAG
TCTCCCTGAGCGCTTTCATGAACACAAAAGTGTCAAGTCGAGGAAGCGGAGGTAT
GAGACTCTAGAAAATAATGATGGCCGTCTAGAA.AAGAAAGTCCACAAA.AGCCTG
GAGAAGGACACGCTAGAGGAGCCAAGGGTGAAGAAGCACAAAAAGTCTAA.AAA
5 GAAAAAGAAGTCCAAAGATAAACACCGGGATCGAGAAAGCAGGCACCAGCAGG
AGTCTGATTTTTCAGGAGCATACTCTGATGCTGACCTCCATAGACACCGGAAGAA
AAAGAAGAAAAAGARAAGGCATTCCAGGAAGTCGGAGGACTTTATAAAGGATGT
TGAGATGCGTTTACCGAAGCTCTCCAGCTACGAGGCCGGCGGCCATTTCCGGAGA
ACAGAGGGCAGCTTTCTCCTGGCTGATGGTCTGCCTGTGGAAGACAGCGGCCCTT
!o TCCGGGAGAAAACGAAGCATTTAAGGATGGAAAGCCGGCCTGACAGATGCCGTC
TGTCGGAGTATGGCCAGGATTCAACATTTTGA
Table 14. Deduced amino acid sequence of coding region of mDUB7
t5 C-terminal potential nuclear localization (as well as targeting) sequences
are
underlined.
MTIVDKTEPSDPSTCQNQPGSCEAVSPEDMDTGSASWGAVSSISDVSSHTLPLGPVPG
AVVYSNSSVPEKSKPSPPKDQVLGDGIAPPQKVLFPSEKICLKWQQSHRVGAGLQNL
3o GNTCFANAALQCLTYTPPLANYMLSHEHSKTCHAEGFCMMCTMQTHITQALSNPGD
VIKPMFVINEMRRIARHFRFGNQEDAHEFLQYTVDAMQKACLNGSNKLDRHTQATT
LV CQIFGGYLRSRVKCLNCKGV SDTFDPYLDITLEII~AAQS V TKALEQFVKPEQLD GE
NSYKCSKCKKMVPASKRFTIHRSSNVLTISLKRFANFTGGKIAKDVKYPEYLDIRPYM
SQPNGEPIIYVLYAVLVHTGFNCHAGHYFCYIKASNGLWYQMNDSIVSTSDIRAVLNQ
QAYVLFYIRSHDVKNGGESAHPAHSPGQSSPRPGVSQRVVNNKQVAPGFIGPQLPSH
VMKNTPHLNGTTPVKDTPSSSVSSPNGNTSVNRASPATASTSVQNWSVTRPSVIPDHP
KKQKITISIF3NKLPARQGQAPLNNSLHGPCLEAPSKAAPSSTITNPSAIQSTSNVPTTSTS
P SEACPKPMVNGKAKV GAS VLVPYGAES SEESDEESKGLAKENGVDMMAGTHSDRP
EAAADDGAEASSHELQEPVLLNGANSADSDSQENSLAFDSASCQVQPELHTENLFSK
LNGLPGKVTPAPLQSVPEDRILETFKLTNQAKGPAGEESWTTTGGSSPKDPVSQLEPIS
DEPSPLEIPEAVTNGSTQTPSTTSPLEPTISCTKEDSSVVVSAEPVEGLPSVPALCNSTGT
ILGDTPVPELCDPGDLTANPSQPTEAVKGDTAEKAQDSAMAEVVERLSPAPSVLTGD
GCEQKLLLYLSAEGSEETED S SRS SAV SADTMPPKPDRTTTS S CEGAAEQAAGDRGD
GGHVGPKAQEPSPAKEKMSSLRKVDRGHYRSRRERSSSGEHVRDSRPRl'EDHHHKT~
~5 RHCYSRERPKQDRHPTNSYCNGGQHLGHGDR.ASPERRSLSRYSHHHSRIRSGLEQDW
SRYHHLENEHAW VRERFYQDKLRWDKCRYYHDRYTPLYTARDAREWRPLHGREHD
RLVQSGRPYKDSYWGRKGWELQSRGKERPHFNSPREAPSLAVPLERHLQEKAALSV
QDSSHSLPERFHEHKSVKSRKRRYETLENNDGRLEKKVHKSLEKDTLEEPRVKKHKK
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SKI~II~KKSKDKHRDRESRHQQESDFSGAYSDADLuRURKKKKKKKItHSRKSEDFIKD
VEMRLPKLSSYEAGGHFRRTEGSFLLADGLPVEDSGPFREKTKHLRMESRPDRCRLSE
YGQDSTF
Table 15. Nucleotide sequence alignment of hDUB7 and mDUB7
HDUB7 ATGACCATAGTTGACAAAGCTTCTGAATCTTCAGACCCATCAGCCTATCAGAATCAGCCT 60
MDUB7 ATGACCATAGTTGACAAAA---CTGAACCTTCAGACCCATCAACCTGTCAGAACCAGCCT 57
****************** ***** ************** *** ****** ******
HDUB7 GGCAGCTCCGAGGCAGTCTCACCTGGAGACATGGATGCAGGTTCTGCCAGCTGGGGTGCT 120
MDUB7 GGCAGTTGTGAGGCGGTCTCACCTGAAGACATGGACACAGGCTCTGCCAGCTGGGGCGCT 117
***** * ***** ********** ********* **** ************** ***
HDUB7 GTGTCTTCATTGAATGATGTGTCAAATCACACACTTTCTTTAGGACCAGTACCTGGTGCT 180
MDUB7 GTGTCTTCAATAAGTGATGTCTCAAGTCACACACTTCCATTAGGGCCAGTGCCTGGTGCT 177
********* * * ****** **** ********** * ***** ***** *********
HDUB7 GTAGTTTATTCGAGTTCATCTGTACCTGATAAATCAAAACCATCACCACAAAAGGATCAA 240
MDUB7 GTAGTTTATTCTAACTCGTCTGTACCTGAAAAATCAAAGCCATCACCACCAAAGGATCAA 237
*********** * ** *********** ******** ********** **********
HDUB7 GCCCTAGGTGATGGCATCGCTCCTCCACAGAAAGTTCTTTTCCCATCTGAGAAGATTTGT 300
MDUB7 GTCCTAGGTGATGGCATTGCTCCTCCTCAAAAGGTCCTGTTTCCATCTGAAAAGATTTGT 297
* *************** ******** ** ** ** ** ** ******** *********
HDUB7 CTTAAGTGGCAACAAACTCATAGAGTTGGAGCTGGGCTCCAGAATTTGGGCAATACCTGT 360
MDUB7 CTTAAGTGGCAACAAAGTCATCGAGTTGGCGCTGGGCTCCAGAATTTGGGCAACACCTGT 357
**************** **** ******* *********************** ******
HDUB7 TTTGCCAATGCAGCACTGCAGTGTTTAACCTACACACCACCTCTTGCCAATTACATGCTA 420
MDUB7 TTTGCCAATGCCGCATTGCAGTGTCTGACTTACACGCCACCCCTCGCCAATTACATGTTA 417
*********** *** ******** * ** ***** ***** ** ************ **
HDUB7 TCACATGAACACTCCAAAACATGTCATGCAGAAGGCTTTTGTATGATGTGTACAATGCAA 480
MDUB7 TCCCATGAACACTCCAAGACATGCCACGCAGAAGGATTTTGTATGATGTGCACGATGCAG 477
** ************** ***** ** ******** ************** ** *****
HDUB7 GCACATATTACCCAGGCACTCAGTAATCCTGGGGACGTTATTAAACCAATGTTTGTCATC 540
MDUB7 ACACACATTACCCAGGCACTTAGCAACCCTGGGGATGTTATCAAGCCGATGTTCGTCATC 537
**** ************** ** ** ******** ***** ** ** ***** ******
HDUB7 AATGAGATGCGGCGTATAGCTAGGCACTTCCGTTTTGGAAACCAAGAAGATGCCCATGAA 600
MDUB7 AATGAAATGCGGCGTATAGCTAGACACTTCCGTTTTGGAAACCAAGAAGATGCCCATGAA 597
***** ***************** ************************************
HDUB7 TTCCTTCAATACACTGTTGATGCTATGCAGAAAGCATGCTTGAATGGCAGCAATAAATTA 660
MDUB7 TTTCTTCAGTACACGGTCGATGCCATGCAGAAAGCATGTTTAAATGGCAGCAATAAATTA 657
** ***** ***** ** ***** ************** ** ******************
HDUB7 GACAGACACACCCAGGCCACCACTCTTGTTTGTCAGATATTTGGAGGATACCTAAGATCT 720
MDUB7 GACAGACACACCCAGGCCACCACCCTGGTCTGCCAGATATTTGGAGGCTACCTAAGATCC 717
*********************** ** ** ** ************** ***********
HDUB7 AGAGTCAAATGTTTAAATTGCAAGGGCGTTTCAGATACTTTTGATCCATATCTTGATATA 780
MDUB7 CGAGTTAAATGTTTAAATTGCAAGGGTGTTTCAGATACCTTTGATCCATATCTGGACATA 777
**** ******************** *********** ************** ** ***
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HDUB7 ACATTGGAGATAAAGGCTGCTCAGAGTGTCAACAAGGCATTG~AGCAGTTTGTGAAGCCG 840
MDUB7 ACGTTGGAGATTAAGGCTGCACAGAGTGTTACCAAGGCGTTAGAGCAGTTTGTGAAGCCA 837
** ******** ******** ******** * ****** ** *****************
HDUB7 GAACAGCTTGATGGAGAAAACTCGTACAAGTGCAGCAAGTGTAAAAAGATGGTTCCAGCT 900
MDUB7 GAACAACTGGATGGAGAAAACTCCTACAAGTGCAGCAAGTGCAAAAAAATGGTTCCAGCT 897
***** ** ************** ***************** ***** ************
HDUB7 TCAAAGAGGTTCACTATCCATAGATCCTCTAATGTTCTTACACTTTCTCTGAAACGTTTT 960
MDUB7 TCAAAGAGATTCACAATCCATAGGTCCTCTAATGTTCTTACCATCTCACTGAAGCGCTTT 957
******** ***** ******** ***************** * ** ***** ** ***
HDUB7 GCAAATTTTACCGGTGGAAAAATTGCTAAGGATGTGAAATACCCTGAGTATCTTGATATT 1020
MDUB7 GCCAACTTCACCGGTGGAAAGATTGCTAAGGATGTGAAATATCCTGAGTACCTTGATATC 1017
** ** ** *********** ******************** ******** ********
HDUB7 CGGCCATATATGTCTCAACCCAACGGAGAGCCAATTGTCTACGTCTTGTATGCAGTGCTG 1080
MDUB7 CGGCCCTATATGTCTCAGCCCAATGGAGAGCCAATTATTTATGTTTTGTATGCTGTGCTG 1077
***** *********** ***** ************ * ** ** ******** ******
HDUB7 GTCCACACTGGTTTTAATTGCCATGCTGGCCATTACTTCTGCTACATAAAAGCTAGCAAT 1140
MDUB7 GTGCACACTGGTTTTAATTGTCATGCTGGCCACTACTTTTGCTACATCAAGGCTAGCAAT 1137
** ***************** *********** ***** ******** ** *********
HDUB7 GGCCTCTGGTATCAAATGAATGACTCCATTGTATCTACCAGTGATATTAGATCGGTACTC 1200
MDUB7 GGCCTCTGGTATCAGATGAATGACTCCATCGTGTCCACCAGTGATATCAGAGCAGTGCTT 1197
************** ************** ** ** *********** *** * ** **
HDUB7 AGCCAACAAGCCTATGTGCTCTTTTATATCAGGTCCCATGATGTGAAAAATGGAGGTGAA 1260
MDUB7 AACCAGCAAGCTTACGTGCTCTTTTATATCAGGTCCCATGATGTGAAAAATGGAGGGGAG 1257
* *** ***** ** ***************************************** **
HDUB7 CTTACTCATCCCACCCATAGCCCCGGCCAGTCCTCTCCCCGCCCCGTCATCAGTCAGCGG 1320
MDUB7 TCTGCTCATCCTGCCCATAGCCCCGGCCAATCCTCTCCCCGCCCAGGAGTCAGTCAGCGG 1317
* ******* **************** ************** * ***********
HDUB7 GTTGTCACCAACAAACAGGCTGCGCCAGGCTTTATCGGACCACAGCTTCCCTCTCACATG 1380
MDUB7 GTAGTCAACAACAAGCAGGTGGCTCCAGGGTTTATTGGACCCCAGCTGCCTTCCCATGTG 1377
** **** ****** **** ** ***** ***** ***** ***** ** ** ** **
HDUB7 ATAAAGAATCCACCTCACTTAAATGGGACTGGACCATTGAAAGACACGCCAAGCAGTTCC 1440
MDUB7 ATGAAGAACACGCCACACTTGAATGGCACCACGCCAGTGAAAGACACACCAAGTAGTTCT 1437
** ***** * ** ***** ***** ** *** ********** ***** *****
HDUB7 ATGTCGAGTCCTAACGGGAATTCCAGTGTCAACAGGGCTAGTCCTGTTAATGCTTCAGCT 1500
MDUB7 GTGTCAAGCCCTAACGGAAACACCAGCGTCAATAGGGCCAGTCCTGCTACTGCTTCGACT 1497
**** ** ******** ** **** ***** ***** ******* ** ****** **
HDUB7 TCTGTCCAAAACTGGTCAGTTAATAGGTCCTCAGTGATCCCAGAACATCCTAAGAAACAA 1560
MDUB7 TCTGTGCAGAACTGGTCTGTTACCAGACCCTCAGTTATTCCAGATCACCCCAAGAAACAA 1557
***** ** ******** **** ** ******* ** ***** ** ** *********
HDUB7 AAAATTACAATCAGTATTCACAACAAGTTGCCTGTTCGCCAGTGTCAGTCTCAACCTAA- 1619
MDUB7 AAAATCACCATCAGTATTCACAACAAGTTGCCTGCTCGCCAGGGTCAGGCACCACTGAAT 1617
***** ** ************************* ******* ***** * * ** **
HDUB7 -----CCTTCATAGTAATTCTTTGGAGAACCCTACCAAGCCCGTTCCCTCTTCTACCATT 1674
MDUB7 AACAGCCTCCATGGCCCTTGTCTGGAGGCTCCTAGTAAGGCGGCACCCTCCTCCACCATC 1677
*** *** * ** * ***** **** *** * * ***** ** *****
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HDUB7 ACCAA---TTCTGCAGTACAGTCTACCTCGAACGCATCTACGATGTCAGTTTCTAGTAAA 1731
MDUB7 ACTAACCCTTCTGCAATACAGTCTACCTCGAACGTACCCACAACGTCGACTTC------- 1730
** ** ******* ****************** * * ** * *** ***
HDUB7 GTAACAAAACCGATCCCCCGCAGTGAATCCTGCTCCCAGCCCGTGATGAATGGCAAATCC 1791
MDUB7 -----------------CCCCAGTGAGGCCTGTCCCAAGCCCATGGTGAACGGCAAGGCT 1773
** ****** **** ** ***** ** **** *****
HDUB7 AAGCTGAACTCCAGCGTGCTGGTGCCCTATGGCGCCGAGTCCTCTGAGGACTCTGACGAG 1851
0 MDUB7 AAAGTGGGCGCCAGTGTGCTTGTCCCCTATGGGGCCGAGTCCTCAGAAGAGTCTGATGAG 1833
** ** * **** ***** ** ******** *********** ** ** ***** ***
HDUB7 GAGTCAAAGGGGCTGGGCAAGGAGAATGGGATTGGTACGATTGTGAGCTCCCACTCTCCC 1911
MDUB7 GAGTCGAAGGGCCTGGCCAAGGAGAACGGTGTGGACATGATGGCCGGCACTCACTCCGAT 1893
LS ***** ***** **** ********* ** * * * *** * ** * *****
t0
HDUB7 GGCCAAGA---TGCCGAAGATGAGG------AGGCCACTCCGCACGAGCTTCAAGAACCC 1962
MDUB7 AGGCCAGAAGCTGCTGCAGATGACGGTGCTGAGGCTTCCTCCCATGAGCTTCAAGAACCC 1953
* * *** *** * ****** * **** * * ** *********.******
HDUB7 ATGACCCTAAACGGTGCTAATAGTGCAGACAGCGACAGTGACCCGAAAGAAAACGGCCTA 2022
MDUB7 GTCCTGCTAAATGGTGCTAATAGCGCAGA------CAGTGACTCACAAGAGAACAGCCTG 2007
* ***** *********** ***** ******* * **** *** ****
'S HDUB7 GCGCCTGATGGTGCCAGCTGCCAAGGCCAGCCTGCCCTGCACTCAGAAAATCCCTTTGCT 2082
MDUB7 GCATTTGACAGTGCCAGCTGCCAGGTCCAGCCCGAGCTACACACAGAAAACCTCTTTTCC 2067
** *** ************* * ****** * ** *** ******* * ****
HDUB7 AAGGCAAACGGTCTTCCTGGAAAGTTGATGCCTGCTCCTTTGCTGTCTCTCCCAGAAGAC 2142
30 MDUB7 AAACTTAATGGTCTTCCTGGAAAGGTGACGCCTGCTCCTTTGCAGTCTGTTCCTGAAGAC 2127
** ** *************** *** ************** **** * ** ******
HDUB7 AAAATCTTAGAGACCTTCAGGCTTAGCAACAAACTGAAAGGCTCGACGGATGAAATGAGT 2202
MDUB7 AGAATCCTTGAGACCTTCAAGCTTACCAACCAGGCAAAGGGTCCAGCGGGTGAAGAGAGT 2187
35 * **** * ********** ***** **** * ** ** * *** **** ****
HDUB7 GCACCTGGAGCAGAGAGGGGCCCTCCCGAGGACCGCGACGCCGAGCCTCAGCCTGGCAGC 2262
MDUB7 TGGACTACGACAGGGGGAAGCTCTCCAAAGGACCCTGTTTCACAGCTGGAGCCCATCAGT 2247
** *** * * ** **** ****** * * *** **** ***
HDUB7 CCCGCCGCCGAATCCCTGGAGGAGCCAGATGCGGCCGCCGGCCTCAGCA---GCACCAAG 2319
MDUB7 GATGAGCCCAGTCCCCTTGAGATACCGGAGGCTGTCACCAATGGGAGCACACAGACCCCT 2307
* ** **** *** ** ** ** * * ** **** ***
HDUB7 AAGGCTCCGCCGCCCCGCGATCCCGGCACCCCCGCTACCAAAGAAGGCGCCTGGGAGGCC 2379
MDUB7 TCCACCACATCACCCCTGGAGCCCACCATCAGCTGTACCAAAGAAGACTCGTCCGTTGTT 2367
* * * **** ** *** ** * * *********** * * * * *
HDUB7 ATGGCCGTCGCCCCCGAGGAG-------CCTCCGCCC-----------AGCGCCGGCGAG 2421
MDUB7 GTCTCAGCTGAACCTGTGGAGGGTTTGCCTTCCGTCCCTGCTCTTTGTAACAGCACTGGT 2427
* * * * ** * **** * **** **
HDUB7 GACATCGTGGGGGACACAGCACCCCCTGACCTGTGTGATCCCGGGAGCTTAACAGGCGAT 2481
MDUB7 ACTATCTTGGGGGATACCCCAGTGCCCGAATTGTGTGACCCTGGAGACTTGACTGCCAAC 2487
*** ******* ** ** ** ** ******* ** ** *** **
HDUB7 GCGAGCCCGTTGTCCCAGGACGCAAAGGGGATGATCGCGGAGGGCCCGCGGGACTCGGCG 2541
MDUB7 CCGAGCCAGCCAACCGAAGCAGTGAAAGGTGATACAGCTGAGAAGGCTCAGGACTCTGCC 2547
****** * ** * * * ** ** * ** *** * * ****** **
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HDUB7 TTGGCGGAAGCCCCGGAAGGGTTGAGTCCGGCTCCGCCTGCGCGGTCGGAGGAGCCCTGC 2601
MDUB7 ATGGCTGAAGTGGTGGAGAGGCTGAGCCCTGCTCCCTCAGTACTCACAGGTGACGGGTGT 2607
**** **** *** ** **** ** ***** * * * * * ** **
HDUB7 GAGCAGCCACTCCTTGTTCACCCCAGCGGGGACCACGCCCGGGACGCTCAGGACCCATCC 2661
MDUB7 GAGCAGAAACTCTTACTTTACCTCAGCGCAGAGGGGTCAGAGGAGACAGAAGACTCTTCC 2667
****** **** * ** *** ***** ** * *** * * *** * ***
HDUB7 CAGAGCTTGGGCGCACCCGAGGCCGCAGAGCGGCCGCCAGCTCCTGTGCTGGACATGGCC 2721
MDUB7 AGAAGCTCGGCGGTCTCTGCTGACACGATGC---------CCCCTAAGCCTGACAGGACC 2718
**** ** * * * * * * ** * *** ** **** * **
HDUB7 CCGGCCGGTCACCCGGAAGGGGACGCTGAGCCTAGCCCCGGCGAGAGGGTCGA-GGACGC 2780
MDUB7 ACCACCAGCTCCTGTGAAGGGGCTGCCGAGCAGGCTGCTGGGGACAGAGGCGATGGAGGC 2778
* ** * * ******* ** **** * ** ** ** * *** *** **
HDUB7 C--GCGGCGCCGAAAGCCCCAGGCCCTTCCCCAGCGAAGGAGAAAATCGGCAGCCTCAGA 2838
MDUB7 CATGTGGGACCCAAAGCTCAGGAGCCTTCCCCAGCCAAGGAAAAGATGAGCAGCCTCCGG 2838
* * ** ** ***** * * *********** ***** ** ** ********
HDUB7 AAGGTGGACCGAGGCCACTACCGCAGCCGGAGAGAGCGCTCGTCCAGCGGGGAGCCCGCC 2898
MDUB7 AAAGTGGACCGAGGACACTATCGGAGCCGGAGAGAGCGCTCCTCCAGTGGGGAGCACGTG 2898
** *********** ***** ** ***************** ***** ******* **
HDUB7 AGAGAGAGCAGGAGCAAGACTGAGGGCCACCGTCACCGGCGGCGCCGCACCTGCCCCCGG 2958
MDUB7 AGGGACAGCAGGCCCCGGCCGGAGGACCATCACCATAAGAAGCGGCACTGCTACAGCCGA 2958
** ** ****** * * * **** *** * ** * *** * * ** * ***
HDUB7 GAGCGCGACCGCCAGGACCGCCACGCCCC------------------GGAGCACCACCCC 3000
MDUB7 GAGCGGCCCAAGCAGGACCGACACCCTACTAATTCATACTGCAATGGGGGCCAGCACTTG 3018
***** * ******** *** * * ** ** ***
HDUB7 GGCCACGGCGACAGGCTCAGCCCTGGCGAGCGCCGCTCTCTGGGCAGGTGCAGTCACCAC 3060
MDUB7 GGCCACGGGGACAGAGCCAGCCCT---GAGCGCCGCTCCCTGAGCAGGTATAGTCACCAC 3075
******** ***** ******* *********** *** ****** *********
HDUB7 CACTCCCGACACCGGAGCGGGGTGGAGCTGGACTGGGTCAGACACCACTACACCGAGGGC 3120
MDUB7 CACTCACGGATTAGGAGTGGCCTGGAGCAGGACTGGAGCCGGTACCACCATTTGGAAAAT 3135
***** ** **** ** ****** ******* * * ***** * **
HDUB7 GAGCGTGGCTGGGGCCGGGAGAAGTTCTACCCCGACAGGCCGCGCTGGGACAGGTGCCGG 3180
MDUB7 GAGCATGCTTGGGTCAGGGAGAGATTCTACCAGGACAAGCTGCGGTGGGACAAGTGCAGG 3195
**** ** **** * ****** ******* **** ** *** ******* **** **
HDUB7 TACTACCATGACAGGTACGC---CCTGTACGCTGCCCGGGACT----GGAAGCCCTTCCA 3233
MDUB7 TATTACCACGACAGGTACACGCCCCTATACACGGCCCGGGACGCCCGAGAATGGCGGCCT 3255
** ***** ********* * *** *** * ********* *** * **
HDUB7 CGGC--GGCCGCGAGCACGAGCGGGCCGGGCTGCACGAGCGGCCGCACAAGGACCACAAC 3291
MDUB7 CTGCATGGTCGTGAGCATGACCGCCTTGTCCAGTCTGGACGGCCATACAAGGACAGCTAC 3315
* ** ** ** ***** ** ** * * * * ***** ******** * **
HDUB7 CGGGGCCGTAGGGGCTGCGAGCCGG---CCCGGGAGAGGGAGCGGCACCGCCCCAGCAGC 3348
MDUB7 TGGGGCCGCAAGGGCTGGGAGCTGCAATCCCGGGGGAAGGAACGGCCCCACTTCAACAGC 3375
******* * ****** **** * ****** ** *** **** ** * ** ****
HDUB7 CCCCGCGCAGGCGCGCCCCACGCCCTCGCCCCGCACCCCGACCGCTTCTCCCACGACAGA 3408
MDUB7 CCCCGAGAGG------CCCCTAGCCTTGCTGTGCCCCTCGAGAGACATCTCCAAGAGAAG 3429
***** * * *** *** ** ** ** *** * *** **
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HDUB7 ACTGCACT---TGTAGCCGGAGACAACTGTAACCTCTCTGATCGGTTTCACGAACACGAA 3465
MDUB7 GCTGCGCTGAGTGTGCAGGACAGCAGCCACAGTCTCCCTGAGCGCTTTCATGAACACAAA 3489
**** ** *** * ** * * *** **** ** ***** ****** **
5 HDUB7 AATGGAAAGTCCCGGAAACGGAGACACGACAGTGTGGAGAACAGTGACAGTCATGTTGAA 3525
MDUB7 AGTGTCAAGTCGAGGAAGCGGAGGTATGAGACTCTAGAAAATAATGATGGCCGTCTAGAA 3549
* ** ***** **** ***** * ** * * * ** ** * *** * * * * ***
HDUB7 AAGAAAGCCCGGAGGAGCGAACAGAAGGATCCTCTAGAAGAGCCTAAAGCAAAGAAGCAC 3585
10 MDUB7 AAGAAAGTCCACAAAAGCCTGGAGAAGGACACGCTAGAGGAGCCAAGGGTGAAGAAGCAC 3609
******* ** * *** ******* * ***** ***** * * *********
HDUB7 A.AAAAATCAAAGAAGAAAAAGAAATCCAAAGACAAACACCGAGACCGCGACTCCAGGCAT 3645
MDUB7 AAAAAGTCTAAAAAGAA.AAAGAAGTCCAAAGATAAACACCGGGATCGAGAAAGCAGGCAC 3669
15 ***** ** ** *********** ******** ******** ** ** ** ******
HDUB7 CAGCAGGACTCAGACCTCTCAGCAGCGTGCTCTGACGCTGACCTCCACAGACACAAAAAA 3705
MDUB7 CAGCAGGAGTCTGATTTTTCAGGAGCATACTCTGATGCTGACCTCCATAGACACCGGAAG 3729
******** ** ** * **** *** * ****** *********** ****** **
HDUB7 AAGAAGAAGAAAAAGAAGAGACATTCAAGAAAATCAGAGGACTTTGTTAAAGATTCAGAA 3765
MDUB7 AAAAAGAAGAAAAAGAAAAGGCATTCCAGGAAGTCGGAGGACTTTATAAAGGATGTTGAG 3789
** ************** ** ***** ** ** ** ********* * ** *** **
HDUB7 CTGCACTTACCCAGGGTCACCAGCTTGGAGACTGTCGCCCAGTTCCGGAGAGCCCAGGGT 3825
MDUB7 ATGCGTTTACCGAAGCTCTCCAGCTACGAGGCCGGCGGCCATTTCCGGAGAACAGAGGGC 3849
*** ***** * * ** ****** *** * * ** *** ********* * ****
HDUB7 GGCTTTCCTCTCTCTGGTGGCCCGCCTCTGGAAGGCGTCGGACCTTTCCGTGAGAAAACG 3885
MDUB7 AGCTTTCTCCTGGCTGATGGTCTGCCTGTGGAAGACAGCGGCCCTTTCCGGGAGAAAACG 3909
****** ** *** *** * **** ****** * *** ******** *********
HDUB7 AAACACTTACGGATGGAAAGCAGGGATGACAGGTGTCGTCTCTTTGAGTATGGCCAGGGT 3945
MDUB7 AAGCATTTAAGGATGGAAAGCCGGCCTGACAGATGCCGTCTGTCGGAGTATGGCCAGGAT 3969
** ** *** *********** ** ****** ** ***** * *************
HDUB7 GATTGA------ 3951
MDUB7 TCAACATTTTGA 3981
Table 16. Deduced amino acid sequence alignment of hDUB7 and mDUB7
HDUB7 MTIVDKASESSDPSAYQNQPGSSEAVSPGDMDAGSASWGAVSSLNDVSNHTLSLGPVPGA 60
MDUB7 MTIVDKT-EPSDPSTCQNQPGSCEAVSPEDMDTGSASWGAVSSISDVSSHTLPLGPVPGA 59
******. *,****. ******,***** ***.**********..***,***,*******
HDUB7 VVYSSSSVPDKSKPSPQKDQALGDGIAPPQKVLFPSEKICLKWQQTHRVGAGLQNLGNTC 120
MDUB7 VVYSNSSVPEKSKPSPPKDQVLGDGIAPPQKVLFPSEKICLKWQQSHRVGAGLQNLGNTC 119
**** ****.****** *** ************************.**************
HDUB7 FANAALQCLTYTPPLANYMLSHEHSKTCHAEGFCMMCTMQAHITQALSNPGDVIKPMFVI 180
MDUB7 FANAALQCLTYTPPLANYMLSHEHSKTCHAEGFCMMCTMQTHITQALSNPGDVIKPMFVI 179
****************************************;*******************
HDUB7 NEMRRIARHFRFGNQEDAHEFLQYTVDAMQKACLNGSNKLDRHTQATTLVCQIFGGYLRS 240
MDUB7 NEMRRIARHFRFGNQEDAHEFLQYTVDAMQKACLNGSNKLDRHTQATTLVCQIFGGYLRS 239
*************************************************.***********
HDUB7 RVKCLNCKGVSDTFDPYLDITLEIKAAQSVNKALEQFVKPEQLDGENSYKCSKCKKMVPA 300
MDUB7 RVKCLNCKGVSDTFDPYLDITLEIKAAQSVTKALEQFVKPEQLDGENSYKCSKCKKMVPA 299
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******************************_*****************************
HDUB7 SKRFTIHRSSNVLTLSLKRFANFTGGKIAKDVKYPEYLDIRPYMSQPNGEPIVYVLYAVL 360
MDUB7 SKRFTIHRSSNVLTISLKRFANFTGGKIAKDVKYPEYLDIRPYMSQPNGEPIIYVLYAVL 359
**************.*************************************;*******
HDUB7 VHTGFNCHAGHYFCYIKASNGLWYQMNDSIVSTSDIRSVLSQQAYVLFYIRSHDVKNGGE 420
MDUB7 VHTGFNCHAGHYFCYIKASNGLWYQMNDSIVSTSDIRAVLNQQAYVLFYIRSHDVKNGGE 419
*************************************,**,*******************
HDUB7 LTHPTHSPGQSSPRPVISQRWTNKQAAPGFIGPQLPSHMIKNPPHLNGTGPLKDTPSSS 480
MDUB7 SAHPAHSPGQSSPRPGVSQRVVNNKQVAPGFIGPQLPSHVMKNTPHLNGTTPVKDTPSSS 479
;**.********** ;*****.***,************.:**,****** *,*******
HDUB7 MSSPNGNSSVNRASPVNASASVQNWSVNRSSVIPEHPKKQKITISIHNKLPVRQCQSQPN 540
MDUB7 VSSPNGNTSVNRASPATASTSVQNWSVTRPSVIPDHPKKQKITISIHNKLPARQGQAPLN 539
******;*******.,**.*******,*.****;****************,** *;
HDUB7 --LHSNSLENPTKPVPSSTITN-SAVQSTSNASTMSVSSKVTKPIPRSESCSQPVMNGKS 597
MDUB7 NSLHGPCLEAPSKAAPSSTITNPSAIQSTSNVPTTSTS--------PSEACPKPMVNGKA 591
**, .** *.* " ******* **.*****,_* *.* **.*,;*.;***.
HDUB7 KLNSSVLVPYGAESSEDSDEESKGLGKENGIGTIVSSHS--PGQDAED-EEATPHELQEP 654
MDUB7 KVGASVLVPYGAESSEESDEESKGLAKENGVDMMAGTHSDRPEAAADDGAEASSHELQEP 651
*..;************.********,****., ...:** * *.* **..******
HDUB7 MTLNGANSADSDSDPKENGLAPDGASCQGQPALHSENPFAKANGLPGKLMPAPLLSLPED 714
MDUB7 VLLNGANSADSDS--QENSLAFDSASCQVQPELHTENLFSKLNGLPGKVTPAPLQSVPED 709
*********** ;**,** *,**** ** **,** *.* ******. **** *.***
HDUB7 KILETFRLSNKLKGSTDEMSAPGAERGPPEDRDAEPQPGSPAAESLEEPDAAA-GLSSTK 773
MDUB7 RILETFKLTNQAKGPAGEESWTTTGGSSPKDPVSQLEPISDEPSPLEIPEAVTNGSTQTP 769
:*****:*:*: **.:.* * . . ..*:* .. :* * ...** *:*.; * :.*
HDUB7 KAPPPRDPGTPATKEGAWEAMAVAPEEPPP------SAGEDIVGDTAPPDLCDPGSLTGD 827
MDUB7 STTSPLEPTISCTKEDSSVWSAEPVEGLPSVPALCNSTGTILGDTPVPELCDPGDLTAN 829
....* :* ..***.: .... * * * .- *.***, *.*****~**,:
HDUB7 ASPLSQDAKGMIAEGPRDSALAEAPEGLSPAPPARSEEPCEQPLLVHPSGDHARDAQDPS 887
MDUB7 PSQPTEAVKGDTAEKAQDSAMAEVVERLSPAPSVLTGDGCEQKLLLYLSAEGSEETEDSS 889
.* .. .** ** .:***:**. * *****.. . . *** **;: *.. ....:*.*
HDUB7 QSLGAPEAAERPPAPVLDMAPAGHPEGDAEPSPGERVED-AAAPKAPGPSPAKEKIGSLR 946
MDUB7 RSS-AVSADTMPPKP--DRTTTSSCEGAAEQAAGDRGDGGHVGPKAQEPSPAKEKMSSLR 946
;* * .* ** * * ;... ** ** ;.*;* .. ..*** *******..***
HDUB7 KVDRGHYRSRRERSSSGEPARESRSKTEGHRHRRRRTCPRERDRQDRHAP------EHHP 1000
MDUB7 KVDRGHYRSRRERSSSGEHVRDSRPRPEDHHHKKRHCYSRERPKQDRHPTNSYCNGGQHL 1006
****************** .*.**,:.*.*.*.:*; .*** ;****_. :*
HDUB7 GHGDRLSPGERRSLGRCSHHHSRHRSGVELDWVRHHYTEGERGWGREKFYPDRPRWDRCR 1060
MDUB7 GHGDRASP-ERRSLSRYSHHHSRIRSGLEQDWSRYHHLENEHAWVRERFYQDKLRWDKCR 1065
***** ** *****.* ****** ***;* ** *,*. *,*;.* **.** *; ***;**
HDUB7 YYHDRYA-LYAAR---DWKPFHGGREHERAGLHERPHKDHNRGRRGCEP-ARERERHRPS 1115
MDUB7 YYHDRYTPLYTARDAREWRPLHG-REHDRLVQSGRPYKDSYWGRKGWELQSRGKERPHFN 1124
******. **;** ;*.*,** ***.* **.** **.* * ;* ;** ; .
HDUB7 SPRAGAPHALAPHPDRFSHDRTALVAGDNCN-LSDRFHEHENGKSRKRRHDSVENSDSHV 1174
MDUB7 SPREAP--SLAVPLERHLQEKAALSVQDSSHSLPERFHEHKSVKSRKRRYETLENNDGRL 1182
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*** ,. :** :*. ...:** . *... *.:*****:, ******::.:**.*.:.
HDUB7 EKKARRSEQKDPLEEPKAKKHKKSKKKKKSKDKHRDRDSRHQQDSDLSAACSDADLHRHK 1234
MDUB7 EKKVHKSLEKDTLEEPRVKKHKKSKKKKKSKDKHRDRESRHQQESDFSGAYSDADLHRHR 1242
***..:* :**,****;,*******************.*****.**;*,* ********.
HDUB7 KKKKKKKRHSRKSEDFVKDSELHLPRVTSLETVAQFRRAQGGFPLSGGPPLEGVGPFREK 1294
MDUB7 KKKKKKKRHSRKSEDFIKDVEMRLPKLSSYEAGGHFRRTEGSFLLADGLPVEDSGPFREK 1302
****************;** *.;**.::* *. .:***.:*_* *._* *.*, ******
HDUB7 TKHLRMESRDDRCRLFEYGQGD-- 1316
MDUB7 TKHLRMESRPDRCRLSEYGQDSTF 1326
********* ***** ****
Table 17. Amino acid sequence alignment of catalytic domain among marine DUB1,
DUB2,
hDUB7 and mDUB7. Amino acids that are involved in catalysis in DUBl (Cys-60,
Asp-133,
and His-307) are underlined.
mDUB1 MVVALSFPEADPALSSPDAPELHQDEAQVVEELTVNGKHSLSWESPQGPGCGLQNTGNSC60
mDUB2 MVVSLSFPEADPALSSPGAQQLHQDEAQVWELTANDKPSLSWECPQGPGCGLQNTGNSC60
hDUB7 VVYSSSSVPDKSKPSPQKDQALGDGIAPPQKVLFPSEKICLKWQQTHRVGAGLQNLGNTC120
mDUB7 VVYSNSSVPEKSKPSPPKDQVLGDGIAPPQKVLFPSEKICLKWQQSHRVGAGLQNLGNTC119
:* . * .. *. * :. * * . * .*.*: .. *,**** **:*
mDUBl YLNAALQCLTHTPPLADYMLSQEHSQTCCSPEGCKLCAMEALVTQSLLHSHSGDVMKPSH120
mDUB2 YLNAALQCLTHTPPLADYMLSQEYSQTCCSPEGCKMCAMEAHVTQSLLHSHSGDVMKPSQ120
hDUB7 FANAALQCLTYTPPLANYMLSHEHSKTCHAEGFCMMCTMQAHITQALSN--PGDVIKPMF178
mDUB7 FANAALQCLTYTPPLANYMLSHEHSKTCHAEGFCMMCTMQTHITQALSN--PGDVIKPMF177
********.*****.****;*.*;** : * :*;*;: :**:* : _***;**
mDUB1 ILTSA------FHKHQQE_DAHEFLMFTLETMHESCLQVHRQSKPTSEDSSPIHDIFGGWW174
mDUB2 ILTSA------FHKHQQE_DAHEFLMFTLETMHESCLQVHRQSEPTSEDSSPIHDIFGGLW174
hDUB7 VINEMRRIARHFRFGNQE_DAHEFLQYTVDAMQKACLNGSNKLDRHTQATTLVCQIFGGYL238
mDUB7 VINEMRRIARHFRFGNQEDAHEFLQYTVDAMQKACLNGSNKLDRHTQATTLVCQIFGGYL237
.... *: :******** :*:::*:::**: .. . .. .. . :****
mDUB1 RSQIKCLLCQGTSDTYDRFLDIPLDISSAQSVKQALWDTEKSEELCGDNAYYCGKCRQKM234
mDUB2 RSQIKCLHCQGTSDTYDRFLDVPLDISSAQSVNQALWDTEKSEELRGENAYYCGRCRQKM234
hDUB7 RSRVKCLNCKGVSDTFDPYLDITLEIKAAQSVNKALEQFVKPEQLDGENSYKCSICCKKMV298
mDUB7 RSRVKCLNCKGVSDTFDPYLDITLEIKAAQSVTKALEQFVKPEQLDGENSYKCSKCKKMV297
**:;*** *:*.***;* ;**:.*;*.:****,;** ; *.*:* *,*;*
*.;*:: .
mDUBl PASKTLHVHIAPKVLMWLNRFSAFTGNKLDRKVSYPEFLDLKPYLSEPTGGPLPYALYA294
mDUB2 PASKTLHIHSAPKVLLLVLKRFSAFMGNKLDRKVSYPEFLDLKPYLSQPTGGPLPYALYA294
hDUB7 PASKRFTIHRSSNVLTLSLKRFANFTGGKIAKDVKYPEYLDIRPYMSQPNGEPIVYVLYA358
mDUB7 PASKRFTIHRSSNVLTISLKRFANFTGGKIAKDVKYPEYLDIRPYMSQPNGEPIIYVLYA357
**** : ;* :.:** : *;**; * *,*; ..*.***;**;;**,*;*,*
*, *.***
mDUB1 VLVHDGATSHSG_HYFCCVKAGHGKWYKMDDTKVTRCDVTSVLNENAYVLFYVQQANLKQ352
mDUB2 VLVHEGATCHSG_HYFSYVKARHGAWYKMDDTKVTSCDVTSVLNENAYVLFYVQQTDLKQ352
hDUB7 VLVHTGFNCHAG_HYFCYIKASNGLWYQMNDSIVSTSDIRSVLSQQAYVLFYIRSHDVKN417
mDUB7 VLVHTGFNCHAGHYFCYIKASNGLWYQMNDSIVSTSDIRAVLNQQAYVLFYIRSHDVKN416
**** * ,.*;****, :** ;* **:*:*: *; ,*: ;.**..:******;:.
.:*:
CA 02479647 2004-09-16
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usav2002-0022.sT25.txt
SEQUENCE LISTING
<110> Aventis Pharmaceuticals, Inc.
Hahn, Chang s
Liu, Hong S
<120> HUMAN DEUBIQUITINATING PROTEASE GENE ON CHROMOSOME 7 AND ITS
MURINE ORTHOLOG
<130> USAV2002-0022 Wo PCT
<150> GB 0218518.9
<151> 2002-09-08
<150> us 60/366,601
<151> 2002-03-22
<160> 12
<170> Patentln version 3.2
<210> 1
<211> 19
<212> PRT
<213> Homo Sapiens
<400> 1
Lys Ala Lys Lys His Lys Lys Ser Lys Lys Lys Lys Lys Ser Lys Asp
1 5 10 15
Lys His Arg
<210> 2
<211> 16
<212> PRT
<213> Homo Sapiens
<400> 2
His Arg His Lys Lys Lys Lys Lys Lys Lys Lys Arg His Ser Arg Lys
1 5 10 15
<210> 3
<211> 17
<212> PRT
<213> Homo Sapiens
<400> 3
Lys Lys His Lys Lys Ser Lys Lys Lys Lys Lys Ser Lys Asp Lys His
1 5 10 15
Arg
<210> 4
<211> 16
<212> PRT
<213> Homo Sapiens
<400> 4
Page 1
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His Arg His Arg Lys Lys Lys Lys Lys Lys Lys Arg His Ser Arg Lys
1 5 10 15
<210> 5
<211> 23
<212> DNA
<213> Homo Sapiens
<400> 5 23
ccacgacaga actgcacttg tag
<210> 6
<211> 19
<212> DNA
<213> Homo Sapiens
<400> 6
ccgggacttt ccattttcg 19
<210> 7
<211> 31
<212> DNA
<213> Homo Sapiens
<400> 7
caactgtaac ctctctgatc ggtttcacga a 31
<210>
8
<211>
3951
<212>
DNA
<213> Sapiens
Homo
<400>
8
atgaccatagttgacaaagcttctgaatcttcagacccatcagcctatcagaatcagcct 60
ggcagctccgaggcagtctcacctggagacatggatgcaggttctgccagctggggtgct 120
gtgtcttcattgaatgatgtgtcaaatcacacactttctttaggaccagtacctggtgct 180
gtagtttattcgagttcatctgtacctgataaatcaaaaccatcaccacaaaaggatcaa 240
gccctaggtgatggcatcgctcctccacagaaagttcttttcccatctgagaagatttgt 300
cttaagtggcaacaaactcatagagttggagctgggctccagaatttgggcaatacctgt 360
tttgccaatgcagcactgcagtgtttaacctacacaccacctcttgccaattacatgcta 420
tcacatgaacactccaaaacatgtcatgcagaaggcttttgtatgatgtgtacaatgcaa 480
gcacatattacccaggcactcagtaatcctggggacgttattaaaccaatgtttgtcatc 540
aatgagatgcggcgtatagctaggcacttccgttttggaaaccaagaagatgcccatgaa 600
ttccttcaatacactgttgatgctatgcagaaagcatgcttgaatggcagcaataaatta 660
gacagacacacccaggccaccactcttgtttgtcagatatttggaggatacctaagatct 720
agagtcaaatgtttaaattgcaagggcgtttcagatacttttgatccatatcttgatata 780
acattggagataaaggctgctcagagtgtcaacaaggcattggagcagtttgtgaagccg 840
gaacagcttgatggagaaaactcgtacaagtgcagcaagtgtaaaaagatggttccagct 900
Page 2
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tcaaagaggttcactatccatagatcctctaatgttcttacactttctctgaaacgtttt960
gcaaattttaccggtggaaaaattgctaaggatgtgaaataccctgagta,tcttgatatt1020
cggccatatatgtctcaacccaacggagagccaattgtctacgtcttgtatgcagtgctg1080
gtccacactggttttaattgccatgctggccattacttctgctacataaaagctagcaat1140
ggcctctggtatcaaatgaatgactccattgtatctaccagtgatattagatcggtactc1200
agccaacaagcctatgtgctcttttatatcaggtcccatgatgtgaaaaatggaggtgaa1260
cttactcatcccacccatagccccggccagtcctctccccgccccgtcatcagtcagcgg1320
gttgtcaccaacaaacaggctgcgccaggctttatcggaccacagcttccctctcacatg1380
ataaagaatccacctcacttaaatgggactggaccattgaaagacacgccaagcagttcc1440
atgtcgagtcctaacgggaattccagtgtcaacagggctagtcctgttaatgcttcagct1500
tctgtccaaaactggtcagttaataggtcctcagtgatcccagaacatcctaagaaacaa1560
aaaattacaatcagtattcacaacaagttgcctgttcgccagtgtcagtctcaacctaac1620
cttcatagtaattctttggagaaccctaccaagcccgttccctcttctaccattaccaat1680
tctgcagtacagtctacctcgaacgcatctacgatgtcagtttctagtaaagtaacaaaa1740
ccgatcccccgcagtgaatcctgctcccagcccgtgatgaatggcaaatccaagctgaac1800
tccagcgtgctggtgccctatggcgccgagtcctctgaggactctgacgaggagtcaaag1860
gggctgggcaaggagaatgggattggtacgattgtgagctcccactctcccggccaagat1920
gccgaagatgaggaggccactccgcacgagcttcaagaacccatgaccctaaacggtgct1980
aatagtgcagacagcgacagtgacccgaaagaaaacggcctagcgcctgatggtgccagc2040
tgccaaggccagcctgccctgcactcagaaaatccctttgctaaggcaaacggtcttcct2100
ggaaagttgatgcctgctcctttgctgtctctcccagaagacaaaatcttagagaccttc2160
aggcttagcaacaaactgaaaggctcgacggatgaaatgagtgcacctggagcagagagg2220
ggccctcccgaggaccgcgacgccgagcctcagcctggcagccccgccgccgaatccctg2280
gaggagccagatgcggccgccggcctcagcagcaccaagaaggctccgccgccccgcgat2340
cccggcacccccgctaccaaagaaggcgcctgggaggccatggccgtcgcccccgaggag2400
cctccgcccagcgccggcgaggacatcgtgggggacacagcaccccctgacctgtgtgat2460
cccgggagcttaacaggcgatgcgagcccgttgtcccaggacgcaaaggggatgatcgcg2520
gagggcccgcgggactcggcgttggcggaagccccggaagggttgagtccggctccgcct2580
gcgcggtcggaggagccctgcgagcagccactccttgttcaccccagcggggaccacgcc2640
cgggacgctcaggacccatcccagagcttgggcgcacccgaggccgcagagcggccgcca2700
gctcctgtgctggacatggccccggccggtcacccggaaggggacgctgagcctagcccc2760
ggcgagagggtcgaggacgccgcggcgccgaaagccccaggcccttccccagcgaaggag2820
aaaatcggcagcctcagaaaggtggaccgaggccactaccgcagccggagagagcgctcg2880
tccagcggggagcccgccagagagagcaggagcaagactgagggccaccgtcaccggcgg2940
Page 3
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cgccgcacctgcccccgggagcgcgaccgccaggaccgccacgccccggagcaccacccc3000
ggccacggcgacaggctcagccctggcgagcgccgctctctgggcaggtgcagtcaccac3060
cactcccgacaccggagcggggtggagctggactgggtcagacaccactacaccgagggc3120
gagcgtggctggggccgggagaagttctaccccgacaggccgcgctgggacaggtgccgg3180
tactaccatgacaggtacgccctgtacgctgcccgggactggaagcccttccacggcggc3240
cgcgagcacgagcgggccgggctgcacgagcggccgcacaaggaccacaaccggggccgt3300
aggggctgcgagccggcccgggagagggagcggcaccgccccagcagcccccgcgcaggc3360
gcgccccacgccctcgccccgcaccccgaccgcttctcccacgacagaactgcacttgta3420
gccggagacaactgtaacctctctgatcggtttcacgaacacgaaaatggaaagtcccgg3480
aaacggagacacgacagtgtggagaacagtgacagtcatgttgaaaagaaagcccggagg3540
agcgaacagaaggatcctctagaagagcctaaagcaaagaagcacaaaaaatcaaagaag3600
aaaaagaaatccaaagacaaacaccgagaccgcgactccaggcatcagcaggactcagac3660
ctctcagcagcgtgctctgacgctgacctccacagacacaaaaaaaagaagaagaaaaag3720
aagagacattcaagaaaatcagaggactttgttaaagattcagaactgcacttacccagg3780
gtcaccagcttggagactgtcgcccagttccggagagcccagggtggctttcctctctct3840
ggtggcccgcctctggaaggcgtcggacctttccgtgagaaaacgaaacacttacggatg3900
gaaagcagggatgacaggtgtcgtctctttgagtatggccagggtgattga 3951
<210> 9
<211> 1316
<212> PRT
<213> Homo Sapiens
<400> 9
Met Thr Ile Val Asp Lys Ala Ser Glu Ser Ser Asp Pro Ser Ala Tyr
1 5 10 15
Gln Asn Gln Pro Gly Ser Ser Glu Ala Val Ser Pro Gly Asp Met Asp
ZO 25 30
Ala Gly Ser Ala Ser Trp Gly Ala Val Ser Ser Leu Asn Asp Val Ser
35 40 45
Asn His Thr Leu Ser Leu Gly Pro Val Pro Gly Ala Val Val Tyr Ser
50 55 60
Ser Ser Ser Val Pro Asp Lys Ser Lys Pro Ser Pro Gln Lys Asp Gln
65 70 75 80
Ala Leu Gly Asp Gly Ile Ala Pro Pro Gln Lys Val Leu Phe Pro Ser
85 90 95
Glu Lys Ile Cys Leu Lys Trp Gln Gln Thr His Arg Val Gly Ala Gly
100 105 110
Page 4
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Leu Gln Asn Leu Gly Asn Thr Cys Phe Ala Asn Ala Ala Leu Gln Cys
115 120 125
Leu Thr Tyr Thr Pro Pro Leu Ala Asn Tyr Met Leu Ser His Glu His
130 135 140
Ser Lys Thr Cys His Ala Glu Gly Phe Cys Met Met Cys Thr Met Gln
145 150 155 160
Ala His Ile Thr Gln Ala Leu Ser Asn Pro Gly Asp Val Ile Lys Pro
165 170 175
Met Phe Val Ile Asn Glu Met Arg Arg Ile Ala Arg His Phe Arg Phe
180 185 190
Gly Asn Gln Glu Asp Ala His Glu Phe Leu Gln Tyr Thr Val Asp Ala
195 200 205
Met Gln Lys Ala Cys Leu Asn Gly Ser Asn Lys Leu Asp Arg His Thr
210 215 220
Gln Ala Thr Thr Leu Val Cys Gln Ile Phe Gly Gly Tyr Leu Arg Ser
225 230 235 240
Arg Val Lys Cys Leu Asn Cys Lys Gly Val Ser Asp Thr Phe Asp Pro
245 250 255
Tyr Leu Asp Ile Thr Leu Glu Ile Lys Ala Ala Gln Ser Val Asn Lys
260 265 270
Ala Leu Glu Gln Phe Val Lys Pro Glu Gln Leu Asp Gly Glu Asn Ser
275 280 285
Tyr Lys Cys Ser Lys Cys Lys Lys Met Val Pro Ala Ser Lys Arg Phe
2g0 295 300
Thr Ile His Arg Ser Ser Asn Val Leu Thr Leu Ser Leu Lys Arg Phe
305 310 315 320
Ala Asn Phe Thr Gly Gly Lys Ile Ala Lys Asp Val Lys Tyr Pro Glu
325 330 335
Tyr Leu Asp Ile Arg Pro Tyr Met Ser Gln Pro Asn Gly Glu Pro Ile
340 345 350
Val Tyr Val Leu Tyr Ala Val Leu Val His Thr Gly Phe Asn Cys His
355 360 365
Ala Gly His Tyr Phe Cys Tyr Ile Lys Ala Ser Asn Gly Leu Trp Tyr
370 375 380
Page 5
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Gln Met Asn Asp Ser Ile Val Ser Thr Ser Asp Ile Arg Ser Val Leu
385 390 395 400
Ser Gln Gln Ala Tyr Val Leu Phe Tyr Ile Arg Ser His Asp Val Lys
405 410 415
Asn Gly Gly Glu Leu Thr His Pro Thr His Ser Pro Gly Gln Ser Ser
420 425 430
Pro Arg Pro Val Ile Ser Gln Arg Val Val Thr Asn Lys Gln Ala Ala
435 440 445
Pro Gly Phe Ile Gly Pro Gln Leu Pro Ser His Met Ile Lys Asn Pro
450 455 460
Pro His Leu Asn Gly Thr Gly Pro Leu Lys Asp Thr Pro Ser Ser Ser
465 470 475 480
Met Ser Ser Pro Asn Gly Asn Ser Ser Val Asn Arg Ala Ser Pro Val
485 490 495
Asn Ala Ser Ala Ser Val Gln Asn Trp Ser Val Asn Arg Ser Ser Val
500 505 510
Ile Pro Glu His Pro Lys Lys Gln Lys Ile Thr Ile Ser Ile His Asn
515 520 525
Lys Leu Pro Val Arg Gln Cys Gln Ser Gln Pro Asn Leu His Ser Asn
530 535 540
Ser Leu Glu Asn Pro Thr Lys Pro Val Pro Ser Ser Thr Ile Thr Asn
545 550 555 560
Ser Ala Val Gln Ser Thr Ser Asn Ala Ser Thr Met Ser Val Ser Ser
565 570 575
Lys Val Thr Lys Pro Ile Pro Arg Ser Glu Ser Cys Ser Gln Pro Val
580 585 590
Met Asn Gly Lys Ser Lys Leu Asn Ser Ser Val Leu Val Pro Tyr Gly
595 600 605
Ala Glu Ser Ser Glu Asp Ser Asp Glu Glu Ser Lys Gly Leu Gly Lys
610 615 620
Glu Asn Gly Ile Gly Thr Ile Val Ser Ser His Ser Pro Gly Gln Asp
625 630 635 640
Ala Glu Asp Glu Glu Ala Thr Pro His Glu Leu Gln Glu Pro Met Thr
645 650 655
Page 6
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Leu Asn Gly Ala Asn Ser Ala Asp Ser Asp Ser Asp Pro Lys Glu Asn
660 665 670
Gly Leu Ala Pro Asp Gly Ala Ser Cys Gln Gly Gln Pro Ala Leu His
675 680 685
Ser Glu Asn Pro Phe Ala Lys Ala Asn Gly Leu Pro Gly Lys Leu Met
690 695 700
Pro Ala Pro Leu Leu Ser Leu Pro Glu Asp Lys Ile Leu Glu Thr Phe
705 710 715 720
Arg Leu Ser Asn Lys Leu Lys Gly Ser Thr Asp Glu Met Ser Ala Pro
725 730 735
Gly Ala Glu Arg Gly Pro Pro Glu Asp Arg Asp Ala Glu Pro Gln Pro
740 745 750
Gly Ser Pro Ala Ala Glu Ser Leu Glu Glu Pro Asp Ala Ala Ala Gly
755 760 765
Leu Ser Ser Thr Lys Lys Ala Pro Pro Pro Arg Asp Pro Gly Thr Pro
770 775 780
Ala Thr Lys Glu Gly Ala Trp Glu Ala Met Ala Val Ala Pro Glu Glu
785 790 795 800
,,
Pro Pro Pro Ser Ala Gly Glu Asp Ile Val Gly Asp Thr Ala Pro Pro
805 810 815
Asp Leu Cys Asp Pro Gly Ser Leu Thr Gly Asp Ala Ser Pro Leu Ser
820 825 830
Gln Asp Ala Lys Gly Met Ile Ala Glu Gly Pro Arg Asp Ser Ala Leu
835 840 845
Ala Glu Ala Pro Glu Gly Leu Ser Pro Ala Pro Pro Ala Arg Ser Glu
850 855 860
Glu Pro Cys Glu Gln Pro Leu Leu Val His Pro Ser Gly Asp His Ala
865 870 875 880
Arg Asp Ala Gln Asp Pro Ser Gln Ser Leu Gly Ala Pro Glu Ala Ala
885 890 895
Glu Arg Pro Pro Ala Pro Val Leu Asp Met Ala Pro Ala Gly His Pro
900 905 910
Glu Gly Asp Ala Glu Pro Ser Pro Gly Glu Arg Val Glu Asp Ala Ala
915 920 925
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Ala Pro Lys Ala Pro Gly Pro Ser Pro Ala Lys Glu Lys Ile Gly Ser
930 935 940
Leu Arg Lys Val Asp Arg Gly His Tyr Arg Ser Arg Arg Glu Arg Ser
945 950 955 960
Ser Ser Gly Glu Pro Ala Arg Glu Ser Arg Ser Lys Thr Glu Gly His
965 970 975
Arg His Arg Arg Arg Arg Thr Cys Pro Arg Glu Arg Asp Arg Gln Asp
980 985 990
Arg His Ala Pro Glu His His Pro Gly His Gly Asp Arg Leu Ser Pro
995 1000 1005
Gly Glu Arg Arg Ser Leu Gly Arg Cys Ser His His His Ser Arg
1010 1015 1020
His Arg Ser Gly Val Glu Leu Asp Trp Val Arg His His Tyr Thr
1025 1030 1035
Glu Gly Glu Arg Gly Trp Gly Arg Glu Lys Phe Tyr Pro Asp Arg
1040 1045 1050
Pro Arg Trp Asp Arg Cys Arg Tyr Tyr His Asp Arg Tyr Ala Leu
1055 1060 1065
Tyr Ala Ala Arg Asp Trp Lys Pro Phe His Gly Gly Arg Glu His
1070 1075 1080
Glu Arg Ala Gly Leu His Glu Arg Pro His Lys Asp His Asn Arg
1085 1090 1095
Gly Arg Arg Gly Cys Glu Pro Ala Arg Glu Arg Glu Arg His Arg
1100 1105 1110
Pro Ser Ser Pro Arg Ala Gly Ala Pro His Ala Leu Ala Pro His
1115 1120 1125
Pro Asp Arg Phe Ser His Asp Arg Thr Ala Leu Val Ala Gly Asp
1130 1135 1140
Asn Cys Asn Leu Ser Asp Arg Phe His Glu His Glu Asn Gly Lys
1145 1150 1155
Ser Arg Lys Arg Arg His Asp Ser Val Glu Asn Ser Asp ser His
1160 1165 1170
Val Glu Lys Lys Ala Arg Arg Ser Glu Gln Lys Asp Pro Leu Glu
1175 1180 1185
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Glu Pro Lys Ala Lys Lys His Lys Lys Ser Lys Lys Lys Lys Lys
1190 1195 1200
Ser Lys Asp Lys His Arg Asp Arg Asp Ser Arg His Gln Gln Asp
1205 1210 1215
Ser Asp Leu Ser Ala Ala Cys Ser Asp Ala Asp Leu His Arg His
1220 1225 1230
Lys Lys Lys Lys Lys Lys Lys Lys Arg His Ser Arg Lys Ser Glu
1235 1240 1245
Asp Phe Val Lys Asp Ser Glu Leu His Leu Pro Arg Val Thr Ser
1250 1255 1260
Leu Glu Thr Val Ala Gln Phe Arg Arg Ala Gln Gly Gly Phe Pro
1265 1270 1275
Leu Ser Gly Gly Pro Pro Leu Glu Gly Val Gly Pro Phe Arg Glu
1280 1285 1290
Lys Thr Lys His Leu Arg Met Glu Ser Arg Asp Asp Arg Cys Arg
1295 1300 1305
Leu Phe Glu Tyr Gly Gln Gly Asp
1310 1315
<210>
<211>
2001
<212>
DNA
<213> Sapiens
Homo
<400>
10
gtaaagtctaaactgagaagtggaagtgtgaactggctggaggtggaaggttggaaaaga60
gtcggagaaaagaacagcatgtgcagagcccagagacagcagggacaaaagaaaaaaaaa120
caagacttcagcatggtgggaacgtgacggagagggtgtttggcgaggttattaggtcag180
acaatgtgaagtccagacattaagatgttgtgctgtgggcagttgggccactcctgaaag240
gtgttctttcttcctttccttttctttctttcttttcttgaggcagagtctctctatgtc300
agtctggagtgcagtggcatgatctcggctcactgcaatctctgccttccaggttcaagc360
aattttccttgcctcagcctcccaagtagctgggaatacaggcgtgcgccaccatgcctg420
gttaatttttttatttttagtagagatggggtttccccatgttggccaggctggtctcga480
actcctggactcaagtgatccacccactttggcctcccaaagtgctgggattacaggggt540
gtgagccactgcgccccgcccggccttttttttttttttttttgagacttaatcttgctc600
tgtcaccaaggctggatatcagtggcacggttttggctctctgcaacttctgtctcccag660
gttcaagcgattttcctgactcagcctcccaagtagttgagattacaggtacgtgccacc720
Page 9
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acgcccggctaatttttgtatttttagtagagatgaggtttcactatgttggccagactg780
gtctcaaacgcctgacctcaggtgattcacctgcctcggcctcccaaaatgctgggatta840
caggtgtgcaccaccatgcctgggtaatttttgtttttcgtagagacagggtctcaccat900
gttggccaggctggtctcaaactcctgacctcaagcgatctgcccaccttggcctcccaa960
ggtgctgcaattataggcatgagccaccgcgcccggcctcctgaaaggttttctacatag1020
gagtggcatgtctagatgtggctactgttgggcgattttagaaatatccctaaaagcctt1080
ctgttgacagggtggcataaccagaaggaagcctggctgggaacgctggacctggctctc1140
agtcccagttgctgactggttgcttcattttataggccctggggattctgtctgatctct1200
catacgttctttataaaaattaagttaatgtatgtccagcagttgatgcaatgcccagta1260
catagaaaatgctcaattagtggtagccctaatattttaaaataggactcagaaagaaaa1320
ttataatcaagtcctttcataacagatatttgtgtttgagtttgatatcagtaatggctt1380
acgggttttatttaaaaagtcatacattccatataaatgagcctcttcagaaaaatggtt1440
ttaaaggtgagatctctataattataattttaaaaaatataatgtatttcacttggtgcc1500
atttgcactttaagcacaaaattaagtctagattttttctgtgtagttgatgcttttctc1560
tgaggaattatactcaaattgaagatgtagtcaaatgtattactgtgtataatttttcta1620
gttttaagcagtatagaaggaaaatataggtacttagtaaataaacagaactgagaattg1680
aaatgtccaattataaactgaaatgccagacttttagggggcatgaaatgaaaatgagaa1740
gttcttttaatcaaatacttcactgaagattttaaaataaagattgttgacattcagatt1800
atcatgatgctaaatgtcccaaggggattattacagaaatgttagaaagtactattgttt1860
ttatatttgagtgatgtgtttgaaaatcactttaaaatggctggaatgatcttccaagat1920
ctaacggtagggtaaggagattgcttttctcacctgatgaaacaaatacatacttttcat1980
cttttgcagagttgaacaatg 2001
<210>
11
<211>
3981
<212>
DNA
<213> musculus
Mus
<400>
11
atgaccatagttgacaaaactgaaccttcagacccatcaacctgtcagaaccagcctggc60
agttgtgaggcggtctcacctgaagacatggacacaggctctgccagctggggcgctgtg120
tcttcaataagtgatgtctcaagtcacacacttccattagggccagtgcctggtgctgta180
gtttattctaactcgtctgtacctgaaaaatcaaagccatcaccaccaaaggatcaagtc240
ctaggtgatggcattgctcctcctcaaaaggtcctgtttccatctgaaaagatttgtctt300
aagtggcaacaaagtcatcgagttggcgctgggctccagaatttgggcaacacctgtttt360
gccaatgccgcattgcagtgtctgacttacacgccacccctcgccaattacatgttatcc420
catgaacactccaagacatgccacgcagaaggattttgtatgatgtgcacgatgcagaca480
cacattacccaggcacttagcaaccctggggatgttatcaagccgatgttcgtcatcaat540
Page 10
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gaaatgcggcgtatagctagacacttccgttttggaaaccaagaagatgcccatgaattt600
cttcagtacacggtcgatgccatgcagaaagcatgtttaaatggcagcaataaattagac660
agacacacccaggccaccaccctggtctgccagatatttggaggctacctaagatcccga720
gttaaatgtttaaattgcaagggtgtttcagatacctttgatccatatctggacataacg780
ttggagattaaggctgcacagagtgttaccaaggcgttagagcagtttgtgaagccagaa840
caactggatggagaaaactcctacaagtgcagcaagtgcaaaaaaatggttccagcttca900
aagagattcacaatccataggtcctctaatgttcttaccatctcactgaagcgctttgcc960
aacttcaccggtggaaagattgctaaggatgtgaaatatcctgagtaccttgatatccgg1020
ccctatatgtctcagcccaatggagagccaattatttatgttttgtatgctgtgctggtg1080
cacactggttttaattgtcatgctggccactacttttgctacatcaaggctagcaatggc1140
ctctggtatcagatgaatgactccatcgtgtccaccagtgatatcagagcagtgcttaac1200
cagcaagcttacgtgctcttttatatcaggtcccatgatgtgaaaaatggaggggagtct1260
gctcatcctgcccatagccccggccaatcctctccccgcccaggagtcagtcagcgggta1320
gtcaacaacaagcaggtggctccagggtttattggaccccagctgccttcccatgtgatg1380
aagaacacgccacacttgaatggcaccacgccagtgaaagacacaccaagtagttctgtg1440
tcaagccctaacggaaacaccagcgtcaatagggccagtcctgctactgcttcgacttct1500
gtgcagaactggtctgttaccagaccctcagttattccagatcaccccaagaaacaaaaa1560
atcaccatcagtattcacaacaagttgcctgctcgccagggtcaggcaccactgaataac1620
agcctccatggcccttgtctggaggctcctagtaaggcggcaccctcctccaccatcact1680
aacccttctgcaatacagtctacctcgaacgtacccacaacgtcgacttcccccagtgag1740
gcctgtcccaagcccatggtgaacggcaaggctaaagtgggcgccagtgtgcttgtcccc1800
tatggggccgagtcctcagaagagtctgatgaggagtcgaagggcctggccaaggagaac1860
ggtgtggacatgatggccggcactcactccgataggccagaagctgctgcagatgacggt1920
gctgaggcttcctcccatga,gcttcaagaacccgtcctgctaaatggtgctaatagcgca1980
gacagtgactcacaagagaacagcctggcatttgacagtgccagctgccaggtccagccc2040
gagctacacacagaaaacctcttttccaaacttaatggtcttcctggaaaggtgacgcct2100
gctcctttgcagtctgttcctgaagacagaatccttgagaccttcaagcttaccaaccag2160
gcaaagggtccagcgggtgaagagagttggactacgacagggggaagctctccaaaggac2220
cctgtttcacagctggagcccatcagtgatgagcccagtccccttgagataccggaggct2280
gtcaccaatgggagcacacagaccccttccaccacatcacccctggagcccaccatcagc2340
tgtaccaaagaagactcgtccgttgttgtctcagctgaacctgtggagggtttgccttcc2400
gtccctgctctttgtaacagcactggtactatcttgggggataccccagtgcccgaattg2460
tgtgaccctggagacttgactgccaacccgagccagccaaccgaagcagtgaaaggtgat2520
acagctgagaaggctcaggactctgccatggctgaagtggtggagaggctgagccctgct2580
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ccctcagtactcacaggtgacgggtgtgagcagaaactcttactttacctcagcgcagag2640
gggtcagaggagacagaagactcttccagaagctcggcggtctctgctgacacgatgccc2700
cctaagcctgacaggaccaccaccagctcctgtgaaggggctgccgagcaggctgctggg2760
gacagaggcgatggaggccatgtgggacccaaagctcaggagccttccccagccaaggaa2820
aagatgagcagcctccggaaagtggaccgaggacactatcggagccggagagagcgctcc2880
tccagtggggagcacgtgagggacagcaggccccggccggaggaccatcaccataagaag2940
cggcactgctacagccgagagcggcccaagcaggaccgacaccctactaattcatactgc3000
aatgggggccagcacttgggccacggggacagagccagccctgagcgccgctccctgagc3060
aggtatagtcaccaccactcacggattaggagtggcctggagcaggactggagccggtac3120
caccatttggaaaatgagcatgcttgggtcagggagagattctaccaggacaagctgcgg3180
tgggacaagtgcaggtattaccacgacaggtacacgcccctatacacggcccgggacgcc3240
cgagaatggcggcctctgcatggtcgtgagcatgaccgccttgtccagtctggacggcca3300
tacaaggacagctactggggccgcaagggctgggagctgcaatcccgggggaaggaacgg3360
ccccacttcaacagcccccgagaggcccctagccttgctgtgcccctcgagagacatctc3420
caagagaaggctgcgctgagtgtgcaggacagcagccacagtctccctgagcgctttcat3480
gaacacaaaagtgtcaagtcgaggaagcggaggtatgagactctagaaaataatgatggc3540
cgtctagaaaagaaagtccacaaaagcctggagaaggacacgctagaggagccaagggtg3600
aagaagcacaaaaagtctaaaaagaaaaagaagtccaaagataaacaccgggatcgagaa3660
agcaggcaccagcaggagtctgatttttcaggagcatactctgatgctgacctccataga3720
caccggaagaaaaagaagaaaaagaaaaggcattccaggaagtcggaggactttataaag3780
gatgttgagatgcgtttaccgaagctctccagctacgaggccggcggccatttccggaga3840
acagagggcagctttctcctggctgatggtctgcctgtggaagacagcggccctttccgg3900
gagaaaacgaagcatttaaggatggaaagccggcctgacagatgccgtctgtcggagtat3960
ggccaggattcaacattttga 3981
<210> 12
<211> 1326
<212> PRT
<213> Mus musculus
<400> 12
Met Thr Ile Val Asp Lys Thr Glu Pro Ser Asp Pro Ser Thr Cys Gln
1 5 10 15
Asn Gln Pro Gly Ser Cys Glu Ala Val Ser Pro Glu Asp Met Asp Thr
20 25 30
Gly Ser Ala Ser Trp Gly Ala Val Ser Ser Ile Ser Asp Val Ser Ser
35 40 45
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His Thr Leu Pro Leu Gly Pro Val Pro Gly Ala Val Val Tyr Ser Asn
50 55 60
Ser Ser Val Pro Glu Lys Ser Lys Pro Ser Pro Pro Lys Asp Gln Val
65 70 75 80
Leu Gly Asp Gly Ile Ala Pro Pro Gln Lys Val Leu Phe Pro Ser Glu
85 90 95
Lys Ile Cys Leu Lys Trp Gln Gln Ser His Arg Val Gly Ala Gly Leu
100 105 110
Gln Asn Leu Gly Asn Thr Cys Phe Ala Asn Ala Ala Leu Gln Cys Leu
115 120 125
Thr Tyr Thr Pro Pro Leu Ala Asn Tyr Met Leu Ser His Glu His Ser
130 135 140
Lys Thr Cys His Ala Glu Gly Phe Cys Met Met Cys Thr Met Gln Thr
145 150 155 160
His Ile Thr Gln Ala Leu Ser Asn Pro Gly Asp Val Ile Lys Pro Met
165 170 175
Phe Val Ile Asn Glu Met Arg Arg Ile Ala Arg His Phe Arg Phe Gly
180 185 190
Asn Gln Glu Asp Ala His Glu Phe Leu Gln Tyr Thr Val Asp Ala Met
195 200 205
Gln Lys Ala Cys Leu Asn Gly Ser Asn Lys Leu Asp Arg His Thr Gln
210 215 220
Ala Thr Thr Leu Val Cys Gln Ile Phe Gly Gly Tyr Leu Arg Ser Arg
225 230 235 240
Val Lys Cys Leu Asn Cys Lys Gly Val Ser Asp Thr Phe Asp Pro Tyr
245 250 255
Leu Asp Ile Thr Leu Glu Ile Lys Ala Ala Gln Ser Val Thr Lys Ala
260 265 270
Leu Glu Gln Phe Val Lys Pro Glu Gln Leu Asp Gly Glu Asn Ser Tyr
275 280 285
Lys Cys Ser Lys Cys Lys Lys Met Val Pro Ala Ser Lys Arg Phe Thr
290 295 300
Ile His Arg Ser Ser Asn Val Leu Thr Ile Ser Leu Lys Arg Phe Ala
305 310 315 320
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Asn Phe Thr Gly Gly Lys Ile Ala Lys Asp Val Lys Tyr Pro Glu Tyr
325 330 335
Leu Asp Ile Arg Pro Tyr Met Ser Gln Pro Asn Gly Glu Pro Ile Ile
340 345 350
Tyr Val Leu Tyr Ala Val Leu Val His Thr Gly Phe Asn Cys His Ala
355 360 365
Gly His Tyr Phe Cys Tyr Ile Lys Ala Ser Asn Gly Leu Trp Tyr Gln
370 375 380
Met Asn Asp Ser Ile Val Ser Thr Ser Asp Ile Arg Ala Val Leu Asn
385 390 395 400
Gln Gln Ala Tyr Val Leu Phe Tyr Ile Arg Ser His Asp Val Lys Asn
405 410 415
Gly Gly Glu Ser Ala His Pro Ala His Ser Pro Gly Gln Ser Ser Pro
420 425 430
Arg Pro Gly Val Ser Gln Arg Val Val Asn Asn Lys Gln Val Ala Pro
435 440 445
Gly Phe Ile Gly Pro Gln Leu Pro Ser His Val Met Lys Asn Thr Pro
450 455 460
His Leu Asn Gly Thr Thr Pro Val Lys Asp Thr Pro Ser Ser Ser Val
465 470 475 480
Ser Ser Pro Asn Gly Asn Thr Ser Val Asn Arg Ala Ser Pro Ala Thr
485 490 495
Ala Ser Thr Ser val Gln Asn Trp ser Val Thr Arg Pro ser val Ile
500 505 510
Pro Asp His Pro Lys Lys Gln Lys Ile Thr Ile Ser Ile His Asn Lys
515 520 525
Leu Pro Ala Arg Gln Gly Gln Ala Pro Leu Asn Asn Ser Leu His Gly
530 535 540
Pro Cys Leu Glu Ala Pro Ser Lys Ala Ala Pro Ser Ser Thr Ile Thr
545 550 555 560
Asn Pro Ser Ala Ile Gln Ser Thr Ser Asn Val Pro Thr Thr Ser Thr
565 570 575
Ser Pro Ser Glu Ala Cys Pro Lys Pro Met Val Asn Gly Lys Ala Lys
580 585 590
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Val Gly Ala Ser Val Leu Val Pro Tyr Gly Ala Glu Ser Ser Glu Glu
595 600 605
Ser Asp Glu Glu Ser Lys Gly Leu Ala Lys Glu Asn Gly Val Asp Met
610 615 620
Met Ala Gly Thr His Ser Asp Arg Pro Glu Ala Ala Ala Asp Asp Gly
625 630 635 640
Ala Glu Ala Ser Ser His Glu Leu Gln Glu Pro Val Leu Leu Asn Gly
645 650 655
Ala Asn Ser Ala Asp Ser Asp Ser Gln Glu Asn Ser Leu Ala Phe Asp
660 665 670
Ser Ala Ser Cys Gln Val Gln Pro Glu Leu His Thr Glu Asn Leu Phe
675 680 685
Ser Lys Leu Asn Gly Leu Pro Gly Lys Val Thr Pro Ala Pro Leu Gln
690 695 700
Ser Val Pro Glu Asp Arg Ile Leu Glu Thr Phe Lys Leu Thr Asn Gln
705 710 715 720
Ala Lys Gly Pro Ala Gly Glu Glu Ser Trp Thr Thr Thr Gly Gly Ser
725 730 735
Ser Pro Lys Asp Pro Val Ser Gln Leu Glu Pro Ile Ser Asp Glu Pro
740 745 750
Ser Pro Leu Glu Ile Pro Glu Ala Val Thr Asn Gly Ser Thr Gln Thr
755 760 765
Pro Ser Thr Thr Ser Pro Leu Glu Pro Thr Ile Ser Cys Thr Lys Glu
770 775 780
Asp Ser Ser Val Val Val Ser Ala Glu Pro Val Glu Gly Leu Pro Ser
785 790 795 800
Val Pro Ala Leu Cys Asn Ser Thr Gly Thr Ile Leu Gly Asp Thr Pro
805 810 815
Val Pro Glu Leu Cys Asp Pro Gly Asp Leu Thr Ala Asn Pro Ser Gln
820 825 830
Pro Thr Glu Ala Val Lys Gly Asp Thr Ala Glu Lys Ala Gln Asp Ser
835 840 845
Ala Met Ala Glu Val Val Glu Arg Leu Ser Pro Ala Pro Ser Val Leu
850 855 860
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Thr Gly Asp Gly Cys Glu Gln Lys Leu Leu Leu Tyr Leu Ser Ala Glu
865 870 875 880
Gly Ser Glu Glu Thr Glu Asp Ser Ser Arg Ser Ser Ala Val Ser Ala
885 890 895
Asp Thr Met Pro Pro Lys Pro Asp Arg Thr Thr Thr Ser Ser Cys Glu
900 ~ 905 910
Gly Ala Ala Glu Gln Ala Ala Gly Asp Arg Gly Asp Gly Gly His Val
915 920 925
Gly Pro Lys Ala Gln Glu Pro Ser Pro Ala Lys Glu Lys Met Ser Ser
930 935 940
Leu Arg Lys Val Asp Arg Gly His Tyr Arg Ser Arg Arg Glu Arg Ser
945 950 955 960
Ser Ser Gly Glu His Val Arg Asp Ser Arg Pro Arg Pro Glu Asp His
965 970 975
His His Lys Lys Arg His Cys Tyr Ser Arg Glu Arg Pro Lys Gln Asp
980 985 990
Arg His Pro Thr Asn Ser Tyr Cys Asn Gly Gly Gln His Leu Gly His
gg5 1000 1005
Gly Asp Arg Ala Ser Pro Glu Arg Arg Ser Leu Ser Arg Tyr Ser
1010 1015 1020
His His His Ser Arg Ile Arg Ser Gly Leu Glu Gln Asp Trp Ser
1025 1030 1035
Arg Tyr His His Leu Glu Asn Glu His Ala Trp Val Arg Glu Arg
1040 1045 1050
Phe Tyr Gln Asp Lys Leu Arg Trp Asp Lys Cys Arg Tyr Tyr His
1055 1060 1065
Asp Arg Tyr Thr Pro Leu Tyr Thr Ala Arg Asp Ala Arg Glu Trp
1070 1075 1080
Arg Pro Leu His Gly Arg Glu His Asp Arg Leu Val Gln Ser Gly
1085 1090 1095
Arg Pro Tyr Lys Asp Ser Tyr Trp Gly Arg Lys Gly Trp Glu Leu
1100 1105 1110
Gln Ser Arg Gly Lys Glu Arg Pro His Phe Asn Ser Pro Arg Glu
1115 1120 1125
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Ala Pro Ser Leu Ala Val Pro Leu Glu Arg His Leu Gln Glu Lys
1130 1135 1140
Ala Ala Leu Ser Val Gln Asp Ser Ser His Ser Leu Pro Glu Arg
1145 1150 1155
Phe His Glu His Lys Ser Val Lys Ser Arg Lys Arg Arg Tyr Glu
1160 1165 1170
Thr Leu Glu Asn Asn Asp Gly Arg Leu Glu Lys Lys Val His Lys
1175 1180 1185
Ser Leu Glu Lys Asp Thr Leu Glu Glu Pro Arg Val Lys Lys His
1190 1195 1200
Lys Lys Ser Lys Lys Lys Lys Lys Ser Lys Asp Lys His Arg Asp
1205 1210 1215
Arg Glu Ser Arg His Gln Gln Glu Ser Asp Phe Ser Gly Ala Tyr
1220 1225 1230
Ser Asp Ala Asp Leu His Arg His Arg Lys Lys Lys Lys Lys Lys
1235 1240 1245
Lys Arg His Ser Arg Lys Ser Glu Asp Phe Ile Lys Asp Val Glu
1250 1255 1260
Met Arg Leu Pro Lys Leu Ser Ser Tyr Glu Ala Gly Gly His Phe
1265 1270 1275
Arg Arg Thr Glu Gly Ser Phe Leu Leu Ala Asp Gly Leu Pro Val
1280 1285 1290
Glu Asp Ser Gly Pro Phe Arg Glu Lys Thr Lys His Leu Arg Met
1295 1300 1305
Glu Ser Arg Pro Asp Arg Cys Arg Leu Ser Glu Tyr Gly Gln Asp
1310 1315 1320
Ser Thr Phe
1325
Page 17
