Note: Descriptions are shown in the official language in which they were submitted.
CA 02377768 2001-12-18
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METHOD OF IDENTIFYING TOXIC AGENTS USING DIFFERENTIAL
GENE EXPRESSION
FIELD OF THE INVENTION
The invention relates generally to nucleic acids and polypeptides and in
particular to
the identification of toxic agents in liver tissue using differential gene
expression.
BACKGROUND OF THE INVENTION
Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used for
controlling
pain and fever in both adults and children. One type of NSAID agent is
acetaminophen.
Acetaminophen is well tolerated by most individuals and is reported to not
induce side effects
sometimes observed with other commonly used pain relievers.
When high doses of acetaminophen are ingested, however, complications can
result.
One type of complication is liver damage, e.g. pericentral hepatic necrosis.
Liver toxicity
associated with acetaminophen ingestion is thought to occur when this agent is
metabolized in
the liver to N-acetyl-parabenzoquinoneimine, which can be toxic. In most
healthy individuals,
this potentially toxic compound is degraded in the liver by the polypeptide
glutathione.
However, when acetaminophen is ingested at high doses, insufficient amount of
glutathione
may be present to process the N-acetyl-parabenzoquinoneimine. As a result,
liver damage can
result. In severe cases, potentially fatal hepatic necrosis may follow.
Acetaminophen poisoning can be treated by administering an antidote. The
antidote is
preferably administered as soon as possible after poisoning. However, the
antidote may not be
promptly administered because signs of acetaminophen poisoning can be confused
with flu-
like symptoms. Early indications of acetaminophen poisoning can include, e.g.,
nausea, and
loss of appetite, diarrhea, abdominal pain, and vomiting. These flu-like
symptoms are usually
present between 4 to 12 hours after taking the drug. Later indications of
acetaminophen
2~ poisoning can include, e.g., confusion, jaundice, or unconsciousness.
However, by the time
administering an antidote will have little effect.
CA 02377768 2001-12-18
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SUMMARY OF THE INVENTION
The invention is based in part on the discovery that certain nucleic acids are
differentially expressed in liver tissue of animals treated with
acetaminophen. These
differentially expressed nucleic acids include novel sequences and nucleic
acids sequences
that, while previously described, have not heretofore been identified as
acetaminophen
responsive. These differentially expressed nucleic acids can be used to
identify agents that
damage livers, i.e., are hepatotoxic, e.g. pericentral hepatic necrosis. These
nucleic acids can
in addition be used to identify poisoning associated with ingestion of NSAIDS,
e.g.,
acetaminophen, in a subject.
In various aspects, the invention includes methods of method of screening a
test agent
for toxicity, e.g., hepatotoxicity. For example, in one aspect, the invention
provides a method
of identifying a hepatotoxic agent by providing a test cell population
comprising a cell capable
of expressing one or more nucleic acids sequences responsive to acetaminophen,
contacting
the test cell population with the test agent and comparing the expression of
the nucleic acids
sequences in the test cell population to the expression of the nucleic acids
sequences in a
reference cell population. An alteration in expression of the nucleic acids
sequences in the test
cell population compared to the expression of the gene in the reference cell
population
indicates that the test agent is hepatotoxic.
In a further aspect, the invention provides a method of assessing the
hepatotoxicity of a
test agent in a subject. The method includes providing from the subject a cell
population
comprising a cell capable of expressing one or more acetaminophen-responsive
genes, and
comparing the expression of the nucleic acids sequences to the expression of
the nucleic acids
sequences in a reference cell population that includes cells from a subject
whose exposure
status to a hepatotoxic agent is known. An alteration in expression of the in
the test cell
population compared to the expression of the nucleic acids sequences in the
reference cell
population indicates the hepatotoxicity of the test agent in the subject.
In another aspect, the invention provides a method of diagnosing or
determining
susceptibility to hepatotoxicity. The method includes providing from the
subject a cell
population comprising a cell capable of expressing one or more ACETA
responsive genes, and
comparing the expression of the nucleic acids sequences to the expression of
the nucleic acids
2
CA 02377768 2001-12-18
WO 01/02609 PCT/iJS00/40292
sequences in a reference cell population that includes cells from a-subject
not suffering from
hepatotoxcity. An alteration in expression of the in the test cell population
compared to the
expression of the nucleic acids sequences in the reference cell population
indicates subject has
or is susceptible to a hepatotoxcity.
Also provided are novel nucleic acids, as well as their encoded polypeptides,
whose
expression is responsive to the effects of acetaminophen.
Unless otherwise defined, all technical and scientific terms used herein have
the same
meaning as commonly understood by one of ordinary skill in the art to which
this invention
belongs. Although methods and materials similar or equivalent to those
described herein can
be used in the practice or testing of the present invention, suitable methods
and materials are
described below. All publications, patent applications, patents, and other
references
mentioned herein are incorporated by reference in their entirety. In the case
of conflict, the
present specification, including definitions, will control. In addition, the
materials, methods,
and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the
following
detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based in part on the discovery of changes in
expression
patterns of multiple nucleic acid sequences in rat liver cells following
exposure to
acetaminophen.
The differentially expressed nucleic acids were identified by administering
acetaminophen orally to male 10-14 week old Sprague Dawley rats at either an
ED100 dose of
43.3 mg/kg/day for 72 hours, or an LD10 dose of 133.3 mg/kg/day for 24 hours.
Differential
gene expression was evaluated in two studies. In the first study, the control
animals received
canola oil. In the second study, the control animals received 10% ethanol.
After sacrificing
animals at the designated time points, liver tissue was dissected, total RNA
was recovered
from the dissected tissue, and cDNA prepared.
Sequences expressed in different levels in acetaminophen-treated and control
rats were
identified by subjecting the cDNA to GENECALLINGT~1 differential expression
analysis as
CA 02377768 2001-12-18
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described in U. S. Patent No. 5,871,697 and in Shimkets et al., Nature
Biotechnology 17:798-
803 (1999). The contents of these patents and publications are incorporated
herein by
reference in their entirety.
In the first study, over 400 gene fragments were initially found to be
differentially
expressed in rat liver tissue in response to administering acetaminophen.
Genes fragments
whose expression levels were modulated greater than 8-fold were selected for
further analysis.
Differential expression of the gene fragments was confirmed using a unlabeled
oligonucleotide competition assay as described in Shimkets et al., Nature
Biotechnology
17:198-803. Forty-two confirmed sequences whose expression is modulated at
least 8-fold in
the presence of acetaminophen were identified. These 42 nucleic acid sequences
are referred
to herein as ACETA 1-42, and are summarized in Table 1. The row subheading in
Table 1
(e.g., protein production, signal transduction, etc.) provides a functional
classification for the
protein.
Ten sequences identified in the first study (ACETA: 1-10) represent novel
genes.
Eight of these ten genes show some similarity to previously described
sequences. Of these
eight genes, three have > 95% identity, four genes have between 90%- 94%
identity, and one
gene has 81 % identity, to known genes. The remaining novel genes, ACETA 6 and
7, do not
appear to be related to previously described sequences. The remaining
sequences
(ACETA:11-42) have been previously described.
In the second study, 133 confirmed sequences were identified whose expression
is
modulated in response to acetaminophen. These sequences are summarized in
Table 2. Six of
the 133 nucleic acid sequences identified were also identified in the first
study. These
sequences are ACETA 15, 20, 23, 33, 36, 41. The remaining newly identified 127
nucleic acid
sequences are referred to herein as ACETA 43-170.
Eight sequences identified in the second study represent novel genes (ACTEA43-
45,
48-51 and 54). Four sequences (ACETA 46, 47, 52 and 53) represent novel ESTs.
Seven of
the eight genes show some similarity to previously described sequences. Of
these seven genes,
two have > 90% identity, two genes have between 80%- 89% identity, and three
genes have
between 70%- 79% identity to known genes. One gene, ACETA 43 does not appear
to be
4
CA 02377768 2001-12-18
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related to previously described sequences. The remaining sequences (ACETA:55-
170) have
been previously described.
Newly described sequences are presented herein. For some of the novel
sequences
(i.e., ACETA: 1-10 and ACTETA 43-54), a cloned sequence is provided along with
one or
more additional sequence fragments (e.g., ESTs or contigs) which contain
sequences
substantially identical to, the cloned sequence. Also provided for some
sequences is a
consensus sequence that includes a composite sequence assembled from the
cloned fragments
and additional fragments. For a given ACETA sequence, its expression can be
measured using
any of the associated nucleic acid sequences in the methods described herein.
For previously described sequences (ACETA:11-42 and ACETA 55-170) database
accession numbers are provided. This information allows for one of ordinary
skill in the art to
deduce information necessary for detecting and measuring expression of the
ACETA nucleic
acid sequences. For example, primers for PCR amplification can be made based
on the
sequences corresponding to the database accession numbers, and/or on the
sequences disclosed
herein.
The acetaminophen -responsive nucleic acids discussed herein include the
following:
5
CA 02377768 2001-12-18
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Table 1
Acetaminophen
Effect on Transcript
Level
GenBank ACETA
Description of Sequence Acc# LD10 ED100 Assignment SEO ID NO
NOVEL GENES
Novel gene fragment, 370 bp, ~ X83749 ~ N.D. ~ -35X
.81 % S.I. To rat 5S rRNA gene.
~~Novel gene fragment, 81 bp, ~ V01270 ~ +180x ~ +65x
.98 % S.I. To rat genes for . . ; ;
;18S, 5.8S, and 28S ribosomal
~RNAs
_ .___ __ .____ ___ .___ __. _
;Novel gene fragment, 411 by ; V01227 ~ N.D. ~ -10x
.g5% S.I. to rat alpha-tubulin
Novel gene fragment, 108 bp, ' X04701 ~ +4x ~ +3.5x
.90% S.I. to Human
;compliment component C1r ; ; ;
~.____________________________J______.___._J___________1__________L____________
___1___________~
Novel gene fragment, 348 by . U39284 . N.D. . +50x . ' '
'. . . . 5 . 6
;93% S.I. to lambda phage IacZ; ; ; ; ; ;
;translational fusion vector ; ;
..____________ _______________~__________ _t
Rat novel gene fragment, 622
Abp
..__ _
1 Rat novel gene fragment, 143 1 t
~bp
Novel gene fragment, ;~uu t
94% S.I. to rat polymorphic
marker D17UIA4 Sequence
___ __
___+_ __+ ______9 ______t____
, '~___Q ___E_ 12____.~
-~'___+
Novel gene fragment, 290 5x
bp
64310
2x
.90% S.I. to mouse surfeit ; ; ; ;
; ;
;locus protein 4 ; ; ; ; ; ;
l
Novel gene fragment,166 by 100x 10 13
M38083 ~ -100x F
.98% S.I. to human alpha-N- ; ; ; ;
; ;
;acetylgalactosaminidase ; ; ; ;
; ;
CA 02377768 2001-12-18
WO 01/02609 PCT/US00/40292
GenBank ACETA
Description of Seciuence Acc# LD10 ED100 AssignmentSEQ ID
NO
1.:PROTEIN PRODUCTION
1.2.:mRNA TRANSLATION
1.2.1.:RIBOSOMAL PROTEINS
_
~L18 protein '________________i__-X20156_ _i______ _ ___________
~ -_--5x '__i__ _5x ;
__ _____
, , . . . , ,
, , . . . , ,
L24--_____i___X78443 ~_____5X ___+____5X _~_______
___F_____ ____
~~Rib
l
t
i
12
osoma , . ,
pro ,
e
n
, , ,
, , ,
, , , , . ,
!.____________________________a____________~___________1__________L____________
__ _1___________J
1.2.6.:RIBOSOMAL RNAs
_ __ _
;18S rRNA gene '____________ 1___M11188-+50x-~_____ _ ___________
; +300x -;__ - _ 13
_____
possible contaminants
, , . , . , ,
, , . , . , ,
, , . . , , ,
,___ __ ,____ ___. __ ___.______ _,___________.
_____
;Genes for 18S, 5.8S, and 28S ; V01270
; -50x ~- -10x ; 14
ribosomal RNAs.
, , , . ,
possible contaminants
, , . . , , ,
, . . , ,
1.5.:PROTEIN DEGRADATION
1.5.3.:PROTEASOME CO
M
PO
N
ENTS
_ __ _ _____ _ ___________
___ __ _ _____ ; ;
__ -10x; 15
_
___ ___ ___ _
;Proteasome RN3 subunit ; L17127
; N.D. ;
~mRNA, complete cds. ; ; ;
, . . , . , ,
, , , , . . ,
, , , ,
, , , , ,
2.:SIGNAL TRANSDUCTION -
___,____________,_______________________________________________,
2.1.:PEPTIDE HORMONESIGROWTH FACTORSICYTOKINES
2.1.5.:PEPTIDE HORMONE BINDING
PROTEINS
_ _-_35x________16_ ___________
;Fetuin -_____________________1___X63446_ . _____ , ,
__' __-_35x-__1_ , , ,
, , , , . , ,
, , , ,
,
, , . . ,
, , . ,
, , , , ________L_______________1___________~
, , , ,
,._______________________.____J____________J___________1_
2.14.:DNA BINDING PROTEIN
RMONE
RECEPT
O
R
S
2.14.2.:NUCLEAR HO
___ __ _ _____ _ ___________
__ __ _ _____ ;
_ +5x ; 17 , ,
_ . , ,
__ _ . , ,
_ . , ,
~Farnesoid X activated receptor; ~ , ,
U18374 ; +80x . .
, , .
, , , .
, ,
, , , ,
, , , ,
, , , . . , ,
, , , , . , ,
, , , , . , ,
, _ ___,___ __ ,____ ___.___ _ _.______
;Small heterodimer partner ; D86580 ; ______.___________,
; N.D. ; -40x 18
, , , , . , ,
homolog
, , , . . , ,
, . . . , ,
, , , , . ,
, , , , . , ,
, , , , . , ,
,.__________________________...a____________a___________~__________i___________
____a___________u
3.:CELL CYCLE
3.6.:CELL DEATH REGULATION
3.6.2.:APOPTOSIS INH
IB
I
T
IO
N
__ __ _ _____ _ ___________
__ _ _ _____
_ +85x. 19 . ,
__ ,
_
__ _ __ __ _
Testis enhanced gene . X75855 . +70x
.
, . ,
transcript ; ; ; ; ; ;
, , , , . ,
, . , ,
.___________________________..i____________~___________i__________4____________
___J___________i
4.:BASIC METABOLISM
4.1.:LIPID METABOLISM
4.1.1.:FATTY ACID SYNTHESIS
__
~Stearyl-CoA desaturase _____i__-X02585__ _i______ _ ___________
~ ___+3x "_i_ +2x'_, 20 , ,
, _____ , ,
, . , . ,
, , . , , ,
, , . , , ,
, , . . . ,
, , , . . ,
, , , , . , ,
, , , , . , ,
, , , ,
CA 02377768 2001-12-18
WO 01/02609 PCT/US00/40292
GenBank ACETA
Description of Sequence LD10 ED100 Assignment
Acc# SEO ID NO
4.1.3.:KETONE BODY METABOLISM
~3-hydroxy-3-methylglutary~__, __2X_ __N.D.-__;_____ 2 ______
__ ;___M33648 _ __ __ i_ _ ___________
~CoA synthase
1 1 1 1 1 1
1 1 1 I 1 1
4.2.:STEROID
METABOLISM'~____________~___________.__________.._______________.___________.
4.2.1:CHOLESTEROL BIOSYNTHESIS
_ _
Farnesyl pyrophosphate _I _ _ __ _i____ _____
'____ i___M34477 - __ _2xi__3x _ ___________
__ __ 22
~synthetase . . . . ~
1 I . . I 1
1 1 1 1 . 1 1
1 1 1 1 1 1 1
1 1 1 1 . 1 1
1 1 1 1 1 1 I
1 1 1 1 . 1 1
1 1 I 1 1 1 I
!.____________________________a____________~___________1_ _________L_
______________1___________1
4.2.4.: EXCRETION
;Cytochrome P-450(M-1 ~ _' __O____-=10x_~_____ 23 ______
_____ ~___J02657-_ __ ___. _ _ ___________
I 1 1 1 . I 1
1 I I 1 1 1 1
1 1 1 1 1 1
I 1 I I I I
1 ____1___ _ I _ _____ ______I___________1
Estrogen sulfotransferase 1 __I_ 24
; U50204 __1_ +100x
' ~
+200x
~
~(Ste1) mRNA, complete
1 1 . 1 1 1
1 1 . 1 . 1
1 1 . I . 1
1 I . 1 . I
1 1 1 . 1
L._________________._..______J____________.
_________L_______________1___________1
J_________._1_
4.3.:CARBOHYDRATE METABOLISM
4.3.1.:GLYCOLYSIS/GLUCONEOGENESIS
~Aldolase g--____________________M1014g_~ __2x_i__-
N.D.__________25__________________;
_ -_ __
1 1 1 . . 1
1 1 .
1 . . . 1
I 1 . . . 1
1 1 . . 1 1
1.____________________________J____________.
________L_______________J___________J
;Phosphoenolpyruvate ; . N.D.. 26 ;
K03248 .
J___________3_
;
-70x
;
;carboxykinase (GTP) gene
1 1 1 I 1 1
1 1 1 1 1
1 1 1 1 1 1
1 1 1 I I
1 . I I 1
L__..._._.___._.._.._.__.___.J_.__________J___________1_ _______1
4.3.2.:GLYCOGEN MAN 1
I 1
P 1
U _L_______________1___________1
L
A
TION
_ __ _ _ __ _ _____ ______
_ ; __ _ _ _ ___________
_ -50x; -40x; 27
__
_
__
__
;Glycogen phosphorylase
; L10669
muscle isozyme
1 1 1 . 1 I I
1 1 1 . 1 1 I
1 1 . . 1 I 1
1 1 . . 1 1 1
1 1 . . 1
1.____________________________J____________J___________1_ ________1
4.4.:OXIDATIVE PHOSPHORYLATION 1
_L_______________1___________1
4.4.1.:CITRIC ACID CYCL
E
__ _ __
__ ___ __ __ 2x . 2x ; 28 ; ;
;~Succinyl-CoA synthetase .
; J03621
1 . . 1 1 1
;alpha subunit ; ; ; ; ; ;
1 1 1 . 1 1 1
I 1 . . 1 1 I
1 I . . 1 1 1
1 _ 1 1 1 _1________________1___________1
4.4.2. ELECTRON TRANSPO ~
R
T CHA
I
N
_ __ _ _ __ _ _____ ______
__ ; __ _ __ _ ___________
_ -40x; N.D.; 29
_
;Cytochrome oxidase subunits
; J01435
~I,II, III genes ; ; ; ; ;
I I 1 I 1 I
1 . 1 I 1 I
1 _ 1 1 1 . 1
4.5.:AROMATIC AMINO ACID ,___________________________________________________
FAMILY'__
4.5.7.:UREA CYCLE
~Arginase ___________________i___j02720-______ _____ ________ 30 ______
1 ~ ~ ____~ ___ ~ ___________
1 1 1 -2x1 -4x 1 I 1
1 I 1 1 ~ 1 1
1 1
1 1 1 1 ~ 1 1
1 1 1 1 ~ ~ 1
..____________________________~____________.__________~_________.._____________
__.___________.
4.5.8.:METHYL CYCLE
__ _ _
.S-adenosyl-L-homocysteine-; _ _____ _r____ ______________
~___M15185 - __ ____~ ___ _ 31
1 ~ 1 -3x -3x ~
hydrolase
1 1 ~ . 1 1
1 1 1 . . 1
1 1 1 . 1 I
I 1 I ~ 1 1
1 1 1 . . 1
I I 1 1 . . 1
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GenBank ACETA
Description LD10 ED100AssignmentSEO
of ID
Sequence NO
Acc#
4.7.:BIOSYNTHESIS
OF
COFACTORS,
PROSTHETIC
GROUPS,
CARRIERS
4.7.3.:HEME
&
PORPHY
R
I
N
_ __ ___ ______ _ ___________
__ ___ ___ _____ ; ;
_ N.D. ; ; 32
_ ___ -2x
___
_
;Delta-aminolevulinic
acid
;
X04959
;
;dehydrogenase ; ; ; ;
; . . 1
;
1 .
1 . 1
1 .
1 ________________________________________________
.._________________________________________,__
4.9.:
DETOXIFICATION
4.9.3.:HEAVY
ME
TALS
_ __ ___ ______ _ _______
______ ___ ___ _____ ___
___ -3x ~ -2x ~ 1 1
___ 33 t t
_
~Metallothionein-I
(mt-1)
~
J00750
~
1 1
1
.._________________________________________,__
_________,______________________________________,
4.9.5.:HYDROCARBONS
_ ___ __ ___ ______ _ ___________
___ __ __ _____ ; ;
_ N.D. ; ; 34
;Glutathione +1.8x
S-transferase
Ya
;
K01931
.
;subunit ; ; ; ;
;
;
1.____________________________J____________J_______....J-_________
L_______________1___________J
4.11.:METABOLITE
STORAGE/TRANSPORT
PROTEINS
4.11.1.:EXTRACELLULAR
TRANSPORT
4.11.1.2.:LIPIDS
_ _
~Apolipoprotein _-_30x'__ r _____ _________
C-Ilj-_________ i___ ____ ____
~__-X02596-__' ~ -24x ~ 35
_
.
1
1
.____________________________J____________J_____._____1__________L_____________
__1___________~
4.11.1.3.:STERIODS
_ __
;Transferrin'_________________ _+360x
i___X77158 _;__+380x
--~
_
4.11.1.4.:HYDROCARBONS-_________,__
_________________________________________________
Alpha-2 _--30x'__ i ______ _ ___________
a i__-=60x 3~ _____
globulin __
___________
i___M27434
~
-
1
1
1
1 . . 1
1
l____________________________J____________J_____._.___J__________
L_______________1___________J
S.:TISSUE
ARCHITECTURE
5.1.:CYTOSKELETON
5.1.1.:COMPONENTS
5.1.1.1.:MOTOR
ARM
5.2.:EXTRACELLULAR
MATRIX
5.2.1.:COMPONENT
_____ ___ ____ ______ _ ___________
__ ___ __ _____
_ O ; -15x ; 38
;Collagen
alpha
1
type
III
;
AJ005395
;
N-______,__
_________________________________________________
5.2.3.:BREAKDOWN
INH
I
B
IT
I
O
_ +50x ; ; 39 ; ;
_ N.D.
__
_
_
_
;Alpha-1-antitrypsin
mRNA
;
M32247
;
Icom 1 1 1 1
lete __ ___.___.______ _.___________.
cds. ___ _____
1
1
1 P
_____.___
__
.__
~
Contrapsin-like -15x ; ; 40
protease -15x
;
D00751
~
;
.inhibitor
(CPi-21)
_______ ._______________.___________1
6.:EXTRACELLULAR
ENVIRONMENTAL
REGULATION
6.2.:IMMUNE
SYSTEM
6.2.1.:COMPLEMENT
6.2.1.1.:COMPONENTS
_ ___ _ __ __ ______ _ ___________
___ __ _____
_ +400x ~ ~ 41
~pcRC201 +550x
mRNA
for
pre-pro-
~
X52477
~
.complement
C3
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GenBank ACETA
Description of Sequence Acc# LD10 ED100 Assignment SEQ ID NO
CELLULAR ORGANELLE STRUCTURAL INTEGRITY
7.2.:ENDOPLASMIC RETICULUM/GOLGI APPARATUS
7.2.2.:TRANSMEMBRANE PROTEINS
7.4.: PEROXISOME/LYSOSOMEIENDOSOME
9.:UNKNOWN FUNCTION
9.1.:KNOWN GENES
9.1.2.:UNASSOCIATED
L1 retrotransposon "ORF2 -_______U83119 --; ____O ________ _4x ___i ______ 42
______
1 . 1 . 1 . 1
1 . 1 1 1 1 1
1 1 1 1 1
1 1 1 1 1 1
1 1 1 1 1 1 1
1 1 1 1 1 1 1
1 1 1 1 1 1 1
1 1 1 1 1 1
1 1 1 1 1 1 1
N.D. = Not Determined
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TABLE 2.
Acetaminophen
GenBank Effect on ACETA SE ID
Gene Discovered Acc. # Transcript Level Assicinment NO
Novel Genes
Novel gene fragment, 228 N/A -11.7 43 14
by
Novel gene fragment, 571bp.
90% SI to Human beta- N/A 2 44 15
tubulin [X79535] and
16
Novel gene fragment, 619bp,
79% SI to guanylyl N/A -3.5 45 17
cyclase A / atrial natriuretic and
peptide receptor [J05677] 18
EST193210 Normalized rat AA850443-1.6 46 19
ovary
EST223737 Normalized rat AI180006-1.5 47 20
spleen
Novel gene fragment, 138bp.
89% SI to Mouse N/A 4.3 48 21
complement factor H-related
protein [M29009]
Novel gene fragment, 419bp,
76% SI to mouse N/A -2.9 49 22
voltage-gated sodium channel
[AF121804]
Novel gene Fragment,109
by 89% SI to Bacteria -1.6 50 23
binding macrophage receptor
MARCO
Novel gene fragment, 299bp,
73% SI to Chinese N/A -1.6 51 24
hamster glutathione S-transferase
subunit [L20466]
EST197321 Normalized rat AA8935184.4 52 25
liver
EST227997 Normalized rat AI2313093 53 26
embryo
Novel gene fragment, 205bp,
90% SI to mouse C1 N/A 2.2 54 27
inhibitor gene {AF052039]
Previously Described Genes
17-beta hydroxysteroid dehydrogenaseX91234 -4.5 55
type 2
3 alpha-Hydroxysteroid dehydrogenaseD17310 2 56
60 kDa protein and non-specificM62763 4.3 57
lipid transfer protein 0
Adenylate kinase 3 D13062 -2.5 58
Alanine aminotransferase D10354 -2.8 59
Aldehyde dehydrogenase M73714 1.6 60
Aldehyde oxidase male form AF1104781.9 61
(AOX1 )
Alpha albumin X76456 2.3 62
Alpha-1-macroglobulin M77183 4.4 63
Alpha-2u-globulin (S type) M26837 -13.3 64
Alpha-fibrinogen. X86561 1.8 65
Alpha-tocopherol transfer D16339 4.9 66
protein
Apolipoprotein B M27440 8 67
Argininosuccinate lyase D28501 -2.4 68
Beta-2 glycoprotein I X15551 -3.7 69
Beta-alanine oxoglutarate D87839 1.5 70
Beta-galactoside alpha 2,6-sialyltransferaseM18769 3.7 71
Betaine-homocysteine methyltransferaseU96133 2.8 72
(BHMT)
Bile canaliculus domain-specific
membrane J02997 8 73
glycoprotein
C4BP alpha chain protein 250051 3.2 74
Calnexin L18889 3 75
Calreticulin X79327 2.7 76
Canalicular multidrug resistanceX96393 -1.8 77
protein
Carboxyl methyltransferase M26686 2.6 78
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Acetaminophen
GenBank Effect on ACETA SEQ IDID
Gene Discovered Acc. # Transcript Level Assignment NO
~arboxylesterase M20629 -1.6 79
~athepsin B X82396 -2.3 80
:DK109 Y17323 4.5 81
~eruloplasmin L33869 8 82
~hymotrypsin B (chyB) K02298 -6.3 83
~onnexin protein Cx26 X51615 2.4 84
-reactive protein M83176 4.8 85
M33313
~YP2A2 (Testosterone 15-alpha-hydroxylase)/ 2 86
J04187
~YP2B2 (Cytochrome p450e J00720 4 87
Phenobarbital inducible)
K00996
~YP2B2 (Cytochrome p450e / 5 88
Phenobarbital inducible) M37134
~YP2C11 (Cytochrome p450h) J02657 -1.6 23
~YP2C13 (Cytochrome p450g) M32277 1.9 89
CYP2C23 (Arachidonic acid U04733 2.2 90
epoxygenase)
CYP2C23 (Cytochrome p450 X55446 2.2 91
c117)
CYP2C6 K03501 -1.5 92
CYP2D5 (Cytochrome p450 M22329 3.3 93
isozyme CMFIb)
S48325/
CYP2E1 (Diabetes-inducible)M20131 -3.9 94
CYP3A18 (Cytochrome p450/6 X79991 5 95
beta-2)
X07259/
CYP4A1 (Cytochrome p452) M14972 3.3 96
CYP4A2 M57719 2.5 97
CYP4F1 (Hepatic tumor cytochromeM94548 2 98
p450)
CYPBB (Sterol 12alpha-hydroxylaseAB009686-10 99
P450)
Cystathionine gamma-lyase D17370 3 100
Cytosolic 3-hydroxy 3-methylglutarylX52625 3 101
coenzyme A
Cytosolic malate dehydrogenaseAF0937732.3 102
(Mdh)
Cytosolic NADP-dependent L35317 3.5 103
isocitrate dehydrogenase
D-binding protein J03179 -10 104
Delta 4-3-ketosteroid-5-beta-reductaseD17309 3 105
Dihydropyrimidinase D63704 1.5 106
Elongation factor 2 Y07504 2.2 107
Estrogen sulfotransferase U50205 2 108
(Ste2)
Eukaryotic initiation factorL11651 2.9 109
(eIF-5)
GADD45gamma AB0209781.6 110
Gene 33 polypeptide X07266 5 111
Glucose transporter type L28135 -1.8 112
2
Glu-Pro Dipeptide Repeat U40628 -3 113
Glutathione S-transferase M26874 1.5 114
Ya subunit
Glutathione S-transferase S72506 1.6 115
Yc2 subunit
iGlyceraldehyde-3-phosphate-dehydrogenase
I(GAPDH) X02231 1.6 116
Glycogen synthase J05446 -2.5 117
Grp75=75 kda glucose regulatedS78556 3.2 118
protein
Haptoglobin K01933 -4.3 119
Hydroxysteroid sulfotransferaseM31363 1.6 120
12
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Acetaminophen
GenBank Effect on ACETA SEQ IDID
Gene Discovered Acc. # Transcript Level Assignment NO
Interleukin 6 receptor ligandM58587 -3.4 121
binding chain
Lanosterol14-alpha-demethylaseU17697 2.8 122
Leuserpin-2. X74549 2.4 123
L-gulono-gamma-lactone oxidaseJ03536 1.6 124
Lipopolysaccharide binding L32132 -2 125
protein
Lysosomal acid lipase, intracellularS81497 2.2 126
hydrolase
Medium chain acyl-CoA dehydrogenaseJ02791 1.8 127
Metallothionein-i (mt-1 J00750 -6.6 33
)
MHC-associated invariant X13044 -1.7 128
chain gamma
Mitochondria) acetoacetyl-CoAD13921 3.5 129
thiolase
Mitochondria) aldehyde dehydrogenaseX14977 2.8 130
Mitochondria) enoyl-CoA X15958 -1.6 131
hydratase
Mitochondria) succinyl-CoA J03621 2.4 132
synthetase alpha subunit
NAD+-isocitrate dehydrogenase,X74125 -2.6 133
gamma subunit
NADPH-cytochrome P-450 reductaseM12516 -2.2 134
Nucleus-encoded mitochondria)
carbamyl phosphate M12335 3.5 135
synthetase I
Ornithine aminotransferase M11842 1.5 136
Orphan nuclear receptor U20389 -2 137
OR-1
P450 6beta-2 D38381 5 138
PcRC201 pre-pro-complement X52477 3.9 41
C3
Peroxisomal enoyl-CoA: hydratase-3-hydroxyacyl-
CoA bifunctional enzyme K03249 2.5 139
Phospholipid hydroperoxide X82679 -1.6 140
glutathione
PhosphoribosylpyrophosphateD26073 -2.3 141
PhosphoribosylpyrophosphateD26073 -2.3 142
Phosphotidylethanolamine L14441 -1.6 143
N-methyltransferase
PKC-zeta-interacting proteinY08355 -1.8 144
1
Plasma protein Y11283 4.2 145
Plasma proteinase inhibitorJ03552 5 146
alpha-1-inhibitor III
Plasma proteinase inhibitor
alpha-1-inhibitor III groupM22360 5 147
3
Polyprotein 1-microglobulin/bikuninS87544 -3 148
Prealbumin (transthyretin) K03251 4.3 149
Preproalbumin V01222 20 150
Pre-pro-complement C3 X52477 -9 151
Pro alpha 1 collagen type X70369 4 152
III
Proteasome RN3 L17127 -1.6 15
Protein kinase inhibitor 2.1 153
p58
Putative glycogen storage AF080468-1.7 154
disease type 1b protein
rat8srna K01592 -4.4 155
Rat ortholog of M. musculus
mRNA Poly(A) binding 2.4 156
protein, 259 by [X65553]
Rat ortholog of Mus musculus
mRNA for Alix (ALG-2- -1.7 157
interacting protein X),
248bp [AJ005073]
Ribophorin I X05300 2.8 158
Serine protease D88250 1.6 159
Small zinc finger-like proteinAF144701-2 160
(TIM13)
Stearyl-CoA desaturase J02585 10 20
13
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Acetaminophen
GenBank Effect on ACETA SEQ IDID
Gene Discovered Acc. # Transcript Level Assignment NO
Sterol 12-alpha hydroxylaseAB018596-1.7 161
Sulfated glycoprotein 2 X13231 1.5 162
Testosterone 6-beta-hydroxylase,U09725 3.1 163
cytochrome
Transferrin X77158 1.5 36
Trihydroxycoprostanoyl-CoA X95189 -1.7 164
Oxidase
Tumor necrosis factor receptorM63122 -1.5 165
(TNF receptor)
UDP-glucuronosyltransferaseM31109 5.5 166
UDP-glucuronosyltransferase,
Phenobarbital-inducible M13506 4 167
form
UI-R-AAO-wq-a-05-0-ULs1 A1578698-2.6 168
UI-R-AAO
Urinary protein 2 precursorAF1984414.4 169
Very-long-chain acyl-CoA r D851003.1 ~ 170
synthetase
14
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Below follows additional discussion of the novel nucleic acid sequences whose
expression is differentially regulated in the presence of acetaminophen.
S ACETAl
ACETA1 is a novel 370 by gene fragment. The nucleic acid has the following
sequence:
1 GGATCCCAGA CACTGGATGG AATGTTAGAG TTTGATTTTG CTTTTGATTG TGACCGTGCC
1O 61CTGATATTTT TTCCCTCTTG AAGGAAGAAA GTATTTTAGT GGAGCCCACA GTTAAGAGAC
121 TTTGAATTGA AAAAAAGACT TTGAATTTTA AAAGAGATTG GATATTTAAT GGGATTGAAA
181 TTTTAAAATG TAAAGACTGT GGGACTCT=A AAGTTATTTA GACCTTGG~G ATGAATAATA
291 AAGTAAGGGT TGAGGTTTAA TAGTGATGTG GTTGTGTGTC AAATTGACAA GGGGTCAATT
301 GTACTGACTG GCTTTGNGTG TCAACTTA.'-0 TCAANCTGGA GTTNTCNC~.G AAAAAGGAGC
IS 361 CTCAGCCCCC (SEQ ID N0:1?
ACETA2
ACETA2 is a novel 436 by gene fragment. The nucleic acid was initially
identified in a
81 by cloned fragment having the following sequence:
1 TCCCGGGTTA AGGCGCCCGA TGCCG=.CGCT CATCATACCC CAAAAAAGTG TTGGTTGATA
61 TANACAGCAG GACGGGGGCC R (SEQ ID N0:2)
The cloned sequence was assembled into a contig resulting in the following
consensus
sequence:
2 S 1
TGCGGCCGCTCCCGTCCCGTTCCGACTGCCGGCGACGGCCGGGTATGGGCCCGACGCTCCAGCGCCATCCATTTTCAGG
G
81
CTAGTTGATTCGGCAGGTGAGTTGTTr.CACACTCCTTAGCGGATTCCGACTTCCATNGGCCACCGTCCTGCTGTCTAT
AT
161
CAACCAACACCTTTTCTGGGGTATG:yT:,AGCGTCGGCATCGGGCGCCT=AACCCGGCGATTCGGTTCATCCCGCAGC
GCC
241
AGTTCTGCTTACCAAAAGTGGCCCAC_AGGCACTCGCATTCCACGCCCGGCTCCACGCCAGCGAGCCGGGCTTCTTACC
C
321
ATTTAAAGTTTGAGAATAGGTTGAGATCGTTTCGGCCCCAAGACCTCT=.ATCATTCGCTTTACCGGATAAAACTGCGG
GT
3 O 401 TGTCGAGCGACCGCGTTGCCAGAGCGCCAGCTATCCTG (SEQ ID P:0:3)
ACETA3
1S
CA 02377768 2001-12-18
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ACETA3 is a novel 411 by gene fragment. The nucleic acid has the following
sequence:
1
NNCTCNCTTATTTTCCANTTTTTTTCTTCCATNATTCCTTCCCTCAGGGTTTGATGNNGCCCTGAATGTTGATCTGACA
G
S 81
AATTCCAGACCAACCTGGTGCCCTACCCTCGAATCCACTTCCCTCTGGCCACTTATGCCCCTGTCATCTCTGCTGAGAA
A
161
GCCTACCATGAGCAACTTACAGTAGCAGAGATCACCAATGCCTGCTTTGAGNCAGCCAACCAGATGGTGAAATGTGACC
C
241
TCGCCATGGTAAATACATGGCTTGCTGCCTGCTGTACCGTGGTGATGTGGTCCCCAAAGATGTCAATGCTGCCiTTGCC
A
321
CCATCAAGACCAAGCGCACCATCCAGTTTGTGGACTGGTGCCCCACTGGCTTCAAGGTTGGCATTAATTACCAGCCTCC
C
901 ACTGTGGTACC (SEQ ID N0:4)
ACETA4
ACETA4 is a novel 108 by gene fragment. The nucleic acid has the following
sequence:
IS 1 GTGCACCTCA AGTTCCTGGA TCCTTTTGAA ATTGATGACC ACCAGCAAGT ACACTGCCCC
61 TATGACCAGC TCCAGATCTA CGCTAATGGG AAGAACTTGG GTGAATTC (SEQ ID N0:5)
ACETAS
ACETAS is a novel 348 by gene fragment. The nucleic acid has the following
sequence:
1 TGTACACTGCAGCCTCGGTATCCAGCACAACCTGCACOGACAGGCCGGTATATGCCGACA
61 CCTTCTGCGCAAACATCTGGCGGGTTGCGTCCATCCGGGACTGCAGTGTCTCCCCGGACG
121 TCATTCCCGGGAAAAAATGGGCTTGTAGGGGGTTTGGCCCATTCCCACCCTTTATTGGGC
2 S 181 TTGCCCGCTTGTAAAAATTCAANCGGNGGAATTTTCCCACACCCCTNGTTTNTTCCAAGC
241 CGCCANCCACCCGTAAATTTACTGGGGANCCCATTCATI~GAACGCCCGATGGAACCCTNG
301 TTCCGGGGCCGGTNTNNGGTNAACCCNNAANCCCCCCCCCCCCCCCCC
(SEQ
ID
N0:6)
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ACETA6
ACETA6 is a novel 622 by gene fragment. The nucleic acid has the following
sequence:
1
CCTCATCAGCGTGATGAAAGGGAAAGATGTCTTTACCCAAGTTCCCTTGTCTCACGGAGACGGGAF:GACTAACAAGAA
GG
S 81
CTTCTTAACATCGTAAAGACATGAGTTCCGTTGAACTAAGTATATAGGTTGGGTTAGAATGAACGGGTCCGTGTGGGTT
G
161
GTCTAGTGAAGAGATGAGCTAGATGCAATGTGTCATTTAGCGTTATGTCTTTAACCGGTGGGCTGCTGTAAGAATCGGT
G
241
GCAGTTCTCTCTCTGCCGTGTGTTAATTGCTCTGGAACGCTACTAGGACCCGAATACTAAGGCCACATCTCTACGTCTC
T
321
AAGAAGGGGAAATAAGATAGGCTTTAGTCTCACAGTGTGGCCTAGGTGGGGTTGCATGTTACTCCCTAACACGCTACAG
A
901
ATTCAGACATAGTTTCTGTGTGGTGGTAGGTCTGTGCTTTTTTTATCGTCTTGCCTGCCATCTTCCr.GCCAGTGTATG
GT
1O 981
TTATCCAGTCTGTGTGCCAAAGCCAGCCATGTCTCCCACGACCCGTTAGTCCAGAGGAGTTCTGCCCCAACACTAGTTT
C
561 CAGCTGCCCGCTCCTAATGTACACCAATCAAGACAGAATAAAATTTGAGTTGTTCGGTGCA
(SEQ ID N0:7)
ACETA7
1 S ACETA7 is a novel 642 by gene fragment. The nucleic acid was initially
identified in
a 143 by cloned fragment having the following sequence:
1 GGGCCCGAAA AGCAAAGAAC CCCCTGATGC TCCCCGCTGA GACTCACTAG CAGGGTTCCA
61 CGGGGTACGG TCCCCTGCAG TAGATGGGAG GTGGNGGGCA TTGGGAAGGC ACAGACAATC
2 O 121 AAATGTAGAC CGGCTAATAA AGT (SEQ ID N0:8)
The cloned sequence was assembled into a contig resulting in the following
consensus
sequence:
2S 1
NTGTACACTTCGCCCAGTTTCTAAAAGGAAGATTCATTCTATGTGCCCTGACTGCCCGCATCCCGTTGACTTGTCAGCC
C
81
CCAGTGTTCTGGAAGCTGCCACAGAATCACTTGCGAAGTTTAACAGCGAGAACCCCTCAAAACAATATGCACTCGTCAA
A
161
GTCACNCAACG~TACGACCCAGTGGGTAGTTGGTCCTTCTTACTTTGTGGAATATTTGATCAAAGA~TCACCATGTACC
C
241
AGTCTCAGGACAGCTGTTCACTCCAGGCCTCCGACTCTGAGCCCGTTGGTCTTTGCCAAGGTTCACTGATTAAAAGTCC
C
321
GGGGTCCCTCCTCAACGCTTTAAAAAGACTGTCACTGTGTCGTGCGAGTTTTTCGAATCTCAGGACC:-
.GGTCCCTGGAGG
3 O 901
TGAGAACCCTGCTGATACCCAAGATGCTAAGAAACTCCCTCAGAAAAACACAGCCCCTACCAGCTCACCCTCCATAACT
G
981 CACCAAGAGGa-
.."CTATCCAACACCTCCCTGAGCAGGAGGAGCCTGAAGACTCCAAGGGAAAGAGTCCTGAGGAACCCTTT
561
CCTGTGCAGC'IGGATCTAACCACAAACCCACAGGGTGACACACTGGATGTCTCCTTCCTCTACCTGGAGCCTGAGGAA
AA
641
GAAACTGGTGGTCCTGCCTTTCCCTGGGAAGGAACAGCGCTCCCCTGAGTGCCCGGGGCCCGAAAAGCAAAGAACCCCC
T
721
GATGCTCCCCGCTGAGACTCACTAGCAGGGTTCCACGGGGTACGGTCCCCTGCAGTAGATGGGAGGT'GGTGGGCATTG
GG
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801
AAGGCACAGACAATCAAATGTAGACCGGCTAATAAAGTGTGTCCTTTGGATGCTTCTTGATCTCAAAAAAAAAAACTGC
A
881 GATATACNATACATCTNCAGNCCCCCNTT (SEQ ID N0:9)
ACETA8
ACETA8 is a novel 411 by gene fragment. The nucleic acid was initially
identified in
a 308 by cloned fragment having the following sequence:
1 TGATCACACAAGAAAATACAGGGTCTACAGATATCTGTCTCATTCACTCTATCTTACTTG
61 TAGATATTTGGGGAGATTGTAGATNGATAGATAGATNGATAGATAGATAGATAGATAGAT
1O 121 AGATCGATAATAGATAGACGATACCTAGATGGATAGATAGATGATAGATAGATNGATAGA
181 TAGATAGATAGATAGATAGGATAGGGAGATGGAGACAGAGAAGGAGGTGAAGCTGGGGGT
.4i AGAGATAGAGATAGACAGAGATATCTNACATCCTAAACGTGTCTGNTTNATTCTCAATAT
301 TCTGTACA
(SEQ
ID N0:10)
The cloned sequence was assembled into a contig resulting in the following
consensus
sequence:
1
TGTACAGAATATTGAGAATNAANCAGACACGTTTAGGATGTNAGATATCTCTGTCTATCTCTATCTCTACCCCCAGCTT
C
81
ACCTCCTTCTCTGTCTCCATCTCCCTATCCTATCTATCTATCTATCTATCTATCTATCTATCTATCATCTATCTATCCA
T
2O 161
CTAGGTATCTGTCTATCTATTATCGATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTACAATCTCCC
C
241
AAATATCTAAAAGTAAGATAGAGTGAATGAGACAGATATCTGTAGACACTGTATTTTCTTGTGTGATCAGATCTAGTGT
G
321
GTGGATGATAGAAGTTGAACTTGCTTTATTGCTATGTGTTAAAATATTTTGTTTGCATTAAAATGGCCTTTTGAAATGC
T
401 TTTCTGTTCCT (SEQ ID N0:11)
ACETA9
ACETA9 is a novel 290 by gene fragment. The nucleic acid has the following
sequence:
1 YGGCCGAGGATTTCGCCGACCAGTTCCTTCGAGTCACCAAGCAGTACCTGCCTCATGTGG
3 O 61 CACGCCTCTGCCTGATTAGCACCTTCCTGGAAGATGGCATCCGCATGTGGTTCCAGNGGA
121 GNGAGCAGCGCGACTATATTGATACCACCTGGAGCTGGGGTTACCTGCTGGCCTCATCCT
181 TTTCGTGGTTNCCCTCAAACCCCTGCTTGGGGGGAACAAGGNNGGGAAACTGGGCTTGGA
241 GTTTNTTGNANGNTGAAACNGGGGAAANTTTNGGCGNCGGGGGGNGAGNN(SEQ ID
N0:12)
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ACETA10
ACETA10 is a novel 116 by gene fragment. The nucleic acid has the following
sequence:
S 1 TGATCACTGT GAAGTTGGTT TTATCTCGGA GGCCACTGAT GATGTCACCT GAGTAGACGT
61 CCTGGGCCTC ATATATCACA GACCCAGTGA AGTTCAGCTG GCCr.AGGGAG GAGTGGTAGC
121 GATAAGGCAT ATCGGTCCTG CAGCTGAAGA AGACTAAGGC GCTAGC (SEQ ID N0:13)
ACETA43
ACETA43 is a novel 391 by gene fragment. The nucleic acid has the following
sequence:
1
TCTAGAAGTCCTTGGGCCCCAGGGGTCTCTGGGAACTAGCAGCGACACCGTGGAAACACACACAGCTTTGCTGATGGAG
C
81
TGTGGGTACCTCACAGCCCTCAGCCAGAGAAACCAGTCCTCTCTTGCCTGTTGTCCTCCTCGCTGCCACATCGTCTTTG
A
161
GTGACTCCGGAAGATCCCGTTACAGGTAAGATCCCAGGATTTCCAGAACATCTTATCTGCAGTTTCTGTGATTATGCCA
G
IS 241
GAGACACAATTGAGTGCAGACCTTACCAGAACAAGGAGCCACCAAGCTACCATCTCAGGGATAAAATAACTCCCACCAT
T
321
GACTCAAGTTGCTTCAGTTTCTGAAGAACCATAGACTTCCATTGTCTTCCTAGGTTTCCTAAGAAGAATTC
(SEQ ID
N0:19)
ACETA44
ACETA44 is a novel S71 by gene fragment. The nucleic acid was initially
identified in
a 411 by cloned fragment having the following sequence:
1
GGATCCCCAACAATGTGAAGACGGCCGTGTGTGACATCCCTCCTCGTGGCCTCAAGATGTCAGCCACCTTCATTGGCAA
C
81
AGCACCGCCATCCAGGAGCTGTTCAAGCGCATCTCGGAGCAGTTCACTGCCATGTTCAGGCGCAAGGCTTTCCTGCACT
G
161
GTACACGGGCGAGGGCATGGACGAGATGGAGTTCACCGAGGCAGAGAGCAACATGAATGACCTGGTGTCTGAGTACCAG
C
2 S 241
AGTACCAGGATGCCACGGCTGATGAGCAGGGCGAGTTCGAGGAGGAGGAGGGCGAGGATGAGGCTTAAGAACTTCTCGG
A
321
TACATTGTGCACCCTTAGTGAACTTCTGTTGTCCTCCAGCATGGTCTTTCTATTTGTAAATTATGGTGCTCAGTTTGCC
T
901 CTGTCTGAAAT (SEQ ID N0:15)
The cloned sequence was assembled into a contig resulting in the following
consensus
sequence:
_
TTTTTTTTTTTTTTCTTAAACACATGCTTTTTTATTCATATAGATTTCTGAGACAATACTGTAATCTTGAAAGGAGGTT
C
81
ACACTATTACATCAACAGTGAATTTCAGACAGAGGCAAACTGAGCACCATAATTTACAAATAGAAAGACCATGCTGGAG
G
161
ACAACAGAAGTTCACTAAGGGTGCACAATGTATCCGAGAAGTTCTTAAGCCTCATCCTCGCCCTCCTCCTCCTCGAACT
C
19
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291
GCCCTGCTCATCAGCCGTGGCATCCTGGTACTGCTGGTACTCAGACACCAGGTCATTCATGTTGCTCTCTGCCTCGGTG
A
321
ACTCCATCTCGTCCATGCCCTCGCCCGTGTACCAGTGCAGGAAAGCCTTGCGCCTGAACATGGCAGTGAACTGCTCCGA
G
901
ATGCGCTTGAACAGCTCCTGGATGGCGGTGCTGTTGCCAATGAAGGTGGCTGACATCTTGAGGCCACGAGGAGGGATGT
C
481
ACACACGGCCGTCTTCACATTGTTGGGGATCCACTCCACGAAGTANCTTCTGTTCTTGTTCTGCACGTTGAGCATCTGC
T
S 561 CATCCACCTCC (SEQ ID N0:16)
ACETA45
ACETA45 is a novel 619 by gene fragment. The nucleic acid was initially
identified in
a 408 by cloned fragment having the following sequence:
1O 1
GGATCCTATGATCCTGAACGGCAGCCTGTGCTCTCTGTCTACCAGCCAGAGGACAACCTTGGAGGCTCTCCCGAGACTC
C
__ __"GTACTCACCCCTGCTAGTGG.'-
.GAAGGTGG,CAGAGGACC=.GGAGGAGGGCTGAGAGGTTGCTCTGATGCTCAT~.GGTGT
161
A.TCATCAGTGCCTACGTGTCTATCCGGCTACTACCAAGACCCTTCCCAGGGCAAGATATCAGTCTAGCATCTCCTACA
GA
291
GGTATCCAGTCTCTAAGGAGGGGACAGTTTAGAAGACAGGGCAGTGACCCCGAAAACGTGCACAGCCCAGG~~TA_AGA
F-:T
32'_
~TCCATACCAGGGT_GGGGAT'_'"_'AGCTCAG"_'GGGTAGAGCGCTTGCCTAAGGAAGTGCAAGGCCCTGGGTTCG
GTCCCCA
1S 901 GCTCCGGA (SEQ ID N0:1,)
The cloned sequence was assembled into a contig resulting in the following
consensus
sequence:
2 O 1 TTTTTTTTTTTTTTTTTACAT~-
.GGAAAATAGTCTTTATTGGTCTTCTGAAACGACAAACCAGAAATATAATTTTGCCTTT
81
AAAAATCTTCTGTCTCAGGCTAAAGATATCACCACTGACAACACCCTCCCTCCCCCAGGCCCTTAGAAAATCCCGGTTC
T
16i
GGGATTGGCCCACCAGCCAAAAGAGGAAGGAAGGCTGTGGCCCAGCCTAGAGGATCCTATGATCCTGAACGGCAGCCTG
T
291
GCTCTCTGTCTACCAGCCAGA_GGACAACCTTGGAGGCTCTCCCGAGACTCCCTGTACTCACCCCTGCTAGTGGAGAAG
GT
3== GGCAGAGGACCAGGAGGAGGGCTGAGAGGTTGCTCTGATGC
TCATAGGTGTATCATCAGTGCCTACGTGTCTATCCGGCT
2S 901
ACTACCAAGACCCTTCCCAGGGCAAGATATCAGTCTAGCr_TCTCCTACAGAGGTATCCAGTCTCTAAGGi;GGGGACA
GTT
481
TAGAAGACAGGGCAGTGACCCCGAAAACGTGCACAGCCCAGGAGTAAGAATCTCCATACCAGGGTTGGGGATTTAGCTC
A
561 GTGGGTAGAGCGCTTGCCTAAGGAAGTGCAAGGCCCTGGGTTCGGTCCCCAGCTCCGGA
(SEQ ID N0:18)
ACETA46
30 ACETA46 is a novel 492 by EST. The nucleic acid has the following sequence:
1
GGCATAGGATAGCTTCGTCTTAGCTTTCTACAATACTGTGTTTTAAGTCTACCCAGTAAACTAAGAGGCAGTACAACTT
G
81
AATACAACTGAGGAGGTGAGAAGCAACTCGGCAGGCCCTGGTGCTCAACAGTTCCGGAATAAACAGGTAGCGCGCAGCC
G
161
CATTAAAAGGTAGTTTGCAAATATTCAAGTTACACCGAGATTCCTTCCAAACCTCTGCCTTGCTCAAACCAGCGCACCT
G
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241
GCCACTGCTAACACCGCGGCACGGCATTTGGTGAATGGTGATGGTTTTACTTTCTTCTTAATCAAAAGCCTGTGGAGGT
G
321
CTGCCACGCAGCAGAGTAAGTCAGCTCTGCTCGAGGACGAGGTGCCAATTGTGAAGTCACAAAAGGTACAGTTTACATG
A
401
GAAACG~~ACATTAACAAGTGGCCCAAATACAGAGCTGGACAGAACCAGACCAGTANGTCTATAGTTACTCAACTGAAC
TA
s81 ACATAAGTACAT (SEQ ID N0:19)
S
ACETA47
ACETA47 is a novel 413 by EST. The nucleic acid has the following sequence:
1
GAAGGAGTGAATGTCCACTGGAGTTTATTTACAGACAACCTTAGGTAAGGCATTTTCCTCTAGGATCTACATCTTGTGA
A
81
GTTACTTGGCTTCAGGCTTCTTGTCTCCAGCTTCAAGCTTGAGATGCTCAGGGGGCTGACGATAGGCAGGGAAAGCCTG
C
1O 161
CAGGGGCTGTTCAGGTCAAACTTGCGGAACTCTTGTGCCAACTCCACTGGCTAAGCCACTACCGGCTTAACCTGATCGG
C
241
ATAACGTAGCTCAACATAGCCAGTGAGGGGAAAGTCTTTCCGGAAAGGATGTCCCTCGAAGCCATAATCTGTCAGGATC
C
321
TTCTCrvAGTCAGGGTGGTTGAAGAAGAAAACTCCAAACATGTCCCAGACCTCCCTCTCATACCAATTGGCCGTTCCAA
CA
401 TCAGCCTCGTGCC (SEQ ID N0:20)
ACETA48
ACETA48 is a novel 138 by gene fragment. The nucleic acid has the following
sequence:
1
GGGCCCTGAGTTAGGTAACCATCTTGGCAAAGAACAGATACTCTCTCTCCATCCAAGTATTTCACTGTGGTTTCAATCA
C
2 O 81 TTGGGCATTTGGAATCTGTGGGGGAGGAGGGCAGAATCTTTTCTCATTTCTTGTACA (SEQ ID
N0:21)
ACETA49
ACETA49 is a novel 419 by gene fragment. The nucleic acid has the following
sequence:
2 S 1
AGATCTTTTCCAAATCCTCTGGCCATTCCTGATGCTTGGTCTAGAGAATGTGGACCTTGCAGGAGAGCCCCTGAGAATC
T
81
CTCCCCAGAATAGAGCTGCCAGTCTGGCCAGCACTTAGCTTCTCATTGGCTCACCCCTTGTCTTTACAGATGTTTGTTT
G
lol
TTTGT_".'GTTTGTTTTAGCTGTTGTGTTAACATTTCCTACAGAATGCTTCTTGATAGGAGAGTAACAGAGGGCTGCC
TGG
291
GTCTTAGGACAATCCATCTTTACCAGATGCCCCTTGCCAAGCTCCCGAAAGTCTCTTCAGGTAGAGCTAGGCTTCGCCA
T
321
CAAGGC~ACCGTATAAGCTGTGGCTTGAGGCTGCTGCTATCAGGAGGACAGGAGAAACAGGGTGGGGTGGAGTGTCTCA
G
3 O 401 GAGAGCCATTGTGGGTACC (SEQ ID N0:22)
ACETA50
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ACETA50 is a novel 109 by gene fragment. The nucleic acid has the following
sequence:
1
CAATTGCAACCATAATGAAGATGCAGGAGTGGAATGCCGCTGACCTGGGAGCCTGAGAAGTCATCCGTGTGTTCCCAGG
T
81 GTCTTTGGGCACCACCCACATGGAGATCT (SEQ ID N0:23)
ACETA51
ACETA51 is a novel 299 by gene fragment. The nucleic acid has the following
sequence:
1
AGATCTGGTCACCAACAATGAAGCATTGGCCACCCTTGTTCTGGGCCAGAAGAGTTTCAAATGGCTTCAGGTGTCCTGG
A
1O 81
AGCTCCTTCCTATATTGGCCCTTGTCCTCC'_"TACAGATATGGAGATAGTGCCATGCF.ATGCGCCTGAACACGTCTT
CCAG
1c'=
TCCGTCGTTCACCA'_"GTCCACCAGTGCTGCCTCTTGCTGGTCTTTGCCGTAGa:GCCACAGGAAGCCTTGAATGTGC
CTTG
241 CTCCCAAACATCCAAGGTCACCACCTCCTCCTTCCAACTCTGGCCCTGGTCGGCTAGC (SEQ ID N0:24)
ACETA52
ACETA52 is a novel 86 by EST. The nucleic acid has the following sequence:
1
TGTTTGAAGATCGATCTCACTGGCATGGGGAAACATATCTTGCTGCTCCCACTGGGCCTGTCTTTGCTCATGAGCTCCC
T
81 GCTAGC (SEQ ID N0:25)
ACETA53
ACETA53 is a novel 111 by EST. The nucleic acid has the following sequence:
1
GGGCCCTGAACATCATCAATGAAGAGAGAACAGCACTTCAAAGGAGGCCGTTGCAACAGTCCTGTCTCCCTGACCCTGA
G
81 GAAGGACAACTTTTATATGCAAATATGTACA (SEQ ID N0:26)
ACETA54
ACETA54 is a novel 205 by gene fragment. The nucleic acid has the following
sequence:
1
GGGCCCGGGGATGGGATGGACCCCCAGAGCTCCTAGGCTCCGCGCCTGGCTCAGAGGCTAACTGGCTTCGTAGGACGCA
G
81
CTGACATCGCTGCCCAGATGGCCTCCAAGCTGACCCCACTGACCCTCCTCCTGCTGCTGCTAGCTGGGGATAGAGCCTT
C
161 TCAGATTCTGAAGTGACCAGCCACAGCTCCC7GGATCCACTAGT (SEQ ID N0:27)
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GENERAL METHODS
The ACETA nucleic acids and encoded polypeptides can be identified using the
information provided above. In some embodiments, the ACETA nucleic acids and
polypeptide correspond to nucleic acids or polypeptides which include the
various sequences
(referenced by SEQ ID NOs) disclosed for each ACETA polypeptide.
In its various aspects and embodiments, the invention includes providing a
test cell
population which includes at least one cell that is capable of expressing one
or more of the
sequences ACETA I-170. By "capable of expressing" is meant that the gene is
present in an
intact form in the cell and can be expressed. Expression of one, some, or all
of the ACETA
sequences is then detected, if present, and, preferably, measured. Using
sequence information
provided by the database entries for the known sequences, or the sequence
information for the
newly described sequences, expression of the ACETA sequences can be detected
(if present)
and measured using techniques well known to one of ordinary skill in the art.
For example,
sequences within the sequence database entries corresponding to ACETA
sequences, or within
the sequences disclosed herein, can be used to construct probes for detecting
ACETA RNA
sequences in, e.g., northern blot hybridization analyses or methods which
specifically, and,
preferably, quantitatively amplify specific nucleic acid sequences. As another
example, the
sequences can be used to construct primers for specifically amplifying the
ACETA sequences
in, e.g., amplification-based detection methods such as reverse-transcription
based polymerise
chain reaction. When alterations in gene expression are associated with gene
amplification or
deletion, sequence comparisons in test and reference populations can be made
by comparing
relative amounts of the examined DNA sequences in the test and reference cell
populations.
Expression can be also measured at the protein level, i.e., by measuring the
levels of
polypeptides encoded by the gene products described herein. Such methods are
well known in
the art and include, e.g., immunoassays based on antibodies to proteins
encoded by the genes.
Expression level of one or more of the ACETA sequences in the test cell
population is
then compared to expression levels of the sequences in one or more cells from
a reference cell
population. Expression of sequences in test and control populations of cells
can be compared
using any art-recognized method for comparing expression of nucleic acid
sequences. For
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example, expression can be compared using GENECALLING~ methods as described in
US
Patent No. 5,871,697 and in Shimkets et al., Nat. Biotechnol. 17:798-803.
In various embodiments, the expression of one or more sequences encoding genes
of
related function, as listed in Table l, is compared. These functions include,
e.g., "Protein
Production" (such as, ACETA 11-14), "Carbohydrate Metabolism" (ACETA 25-26),
"Steroid
Metabolism" (ACETA 22 and 24), and "Detoxifucation" (ACETA 33-34). In some
embodiments, expression of members of two or more functional families are
compared.
In various other embodiments, the expression of 2, 3, 4, 5, 6, 7,8, 9, 10, 25,
50, 100,
150 or all of the sequences represented by ACETA 1-170 are measured. If
desired, expression
of these sequences can be measured along with other sequences whose expression
is known to
be altered according to one of the herein described parameters or conditions.
The reference cell population includes one or more cells for which the
compared
parameter is known. The compared parameter can be, e.g., hepatotoxic agent
expression
status. By "hepatotoxic agent expression status" is meant that it is known
whether the
reference cell has had contact with a hepatotoxic agent. An example of a
hepatotoxic agent is,
e.g., a nonsteroidal anti-inflammatory drug such as acetaminophen. Whether or
not
comparison of the gene expression profile in the test cell population to the
reference cell
population reveals the presence, or degree, of the measured parameter depends
on the
composition of the reference cell population. For example, if the reference
cell population is
composed of cells that have not been treated with a known hepatotoxic agent, a
similar gene
expression level in the test cell population and a reference cell population
indicates the test
agent is not a hepatotoxic agent. Conversely, if the reference cell population
is made up of
cells that have been treated with a hepatotoxic agent, a similar gene
expression profile between
the test cell population and the reference cell population indicates the test
agent is a
hepatotoxic agent.
In various embodiments, a ACETA sequence in a test cell population is
considered
comparable in expression level to the expression level of the ACETA sequence
if its
expression level varies within a factor of 2.0, 1.5, or 1.0 fold to the level
of the ACETA
transcript in the reference cell population. In various embodiments, a ACETA
sequence in a
test cell population can be considered altered in levels of expression if its
expression level
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varies from the reference cell population by more than 1.0, 1.5, 2.0 or more
fold from the
expression level of the corresponding ACETA sequence in the reference cell
population.
If desired, comparison of differentially expressed sequences between a test
cell
population and a reference cell population can be done with respect to a
control nucleic acid
whose expression is independent of the parameter or condition being measured.
Expression
levels of the control nucleic acid in the test and reference nucleic acid can
be used to normalize
signal levels in the compared populations.
In some embodiments, the test cell population is compared to multiple
reference cell
populations. Each of the multiple reference populations may differ in the
known parameter.
Thus, a test cell population may be compared to a first reference cell
population known to have
been exposed to a hepatotoxic agent, as well as a second reference population
known have not
been exposed to a hepatotoxic agent.
The test cell population that is exposed to, i.e., contacted with, the test
hepatotoxic
agent can be any number of cells, i.e., one or more cells, and can be provided
in vitro, in vivo,
or ex vivo.
In other embodiments, the test cell population can be divided into two or more
subpopulations. The subpopulations can be created by dividing the first
population of cells to
create as identical a subpopulation as possible. This will be suitable, in,
for example, in vitro
or ex vivo screening methods. In some embodiments, various sub populations can
be exposed
to a control agent, and/or a test agent, multiple test agents, or, e.g.,
varying dosages of one or
multiple test agents administered together, or in various combinations.
Preferably, cells in the reference cell population are derived from a tissue
type as
similar as possible to test cell, e.g., liver tissue. In some embodiments, the
control cell is
derived from the same subject as the test cell, e.g., from a region proximal
to the region of
origin of the test cell. In other embodiments, the reference cell population
is derived from a
plurality of cells. For example, the reference cell population can be a
database of expression
patterns from previously tested cells for which one of the herein-described
parameters or
conditions (hepatotoxic agent expression status) is known.
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The test agent can be a compound not previously described or can be a
previously
known compound but which is not known to be a hepatotoxic agent.
The subject is preferably a mammal. The mammal can be, e.g., a human, non-
human
primate, mouse, rat, dog, cat, horse, or cow.
SCREENING FOR TOXIC AGENTS
In one aspect, the invention provides a method of identifying toxic agents,
e.g.,
hepatotoxic agents. The hepatotoxic agent can be identified by providing a
cell population that
includes cells capable of expressing one or more nucleic acid sequences
homologous to those
listed in Table 1. And Table 2 as ACETA 1-170. The sequences need not be
identical to
sequences including ACETA 1-170, as long as the sequence is sufficiently
similar that specific
hybridization can be detected. Preferably, the cell includes sequences that
are identical, or
nearly identical to those identifying the ACETA nucleic acids shown in Table 1
and Table 2.
Expression of the nucleic acid sequences in the test cell population is then
compared to
the expression of the nucleic acid sequences in a reference cell population,
which is a cell
population that has not been exposed to the test agent, or, in some
embodiments, a cell
population exposed the test agent. Comparison can be performed on test and
reference
samples measured concurrently or at temporally distinct times. An example of
the latter is the
use of compiled expression information, e.g., a sequence database, which
assembles
information about expression levels of known sequences following
administration of various
agents. For example, alteration of expression levels following administration
of test agent can
be compared to the expression changes observed in the nucleic acid sequences
following
administration of a control agent, such as acetaminophen.
An alteration in expression of the nucleic acid sequence in the test cell
population
compared to the expression of the nucleic acid sequence in the reference cell
population that
has not been exposed to the test agent indicates the test agent is a
hepatotoxic agent.
The invention also includes a hepatotoxic agent identified according to this
screening
method.
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ASSESSING TOXICITY OF AN AGENT IN A SUBJECT
The differentially expressed ACETA sequences identified herein also allow for
the
hepatotoxicity of a toxic agent to be determined or monitored. In this method,
a test cell
population from a subject is exposed to a test agent, i.e. a hepatotoxic
agent. If desired, test
cell populations can be taken from the subject at various time points before,
during, or after
exposure to the test agent. Expression of one or more of the ACETA sequences,
e.g., ACETA:
1-170, in the cell population is then measured and compared to a reference
cell population
which includes cells whose hepatotoxic agent expression status is known.
Preferably, the
reference cells not been exposed to the test agent.
If the reference cell population contains no cells exposed to the treatment, a
similarity
in expression between ACETA sequences in the test cell population and the
reference cell
population indicates that the treatment is non-hepatotoxic. However, a
difference in
expression between ACETA sequences in the test population and this reference
cell population
indicates the treatment is hepatotoxic.
By "hepatotoxicity" is meant that the agent is damaging or destructive to
liver when
administered to a subject. In some embodiments, hepatotoxicity includes
pericentral hepatic
necrosis.
METHODS OF DIAGNOSING HEPATOTOXICITY
The invention further provides a method of diagnosing hepatotoxicity, in a
subject. In
this method, hepatotoxicity is diagnosed by examining the expression of one or
more ACETA
nucleic acid sequences from a test population of cells from a subject
suspected to have been
exposed to a hepatotoxic agent, e.g. non-steroidal anti-inflammatory drugs.
Expression of one or more of the ACETA nucleic acid sequences, e.g. ACETA: 1-
170,
or any combination of these sequences, is measured in the test cell and
compared to the
expression of the sequences in the reference cell population. The reference
cell population
contains at least one cell whose hepatotoxicity status is known. If the
reference cell population
contains cells that have not been exposed to a hepatotoxic agent, than a
similarity in
expression between ACETA sequences in the test population and the reference
cell population
indicates the subject does not have hepatotoxicity. A difference in expression
between
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ACETA sequences in the test population and the reference cell population
indicates the
reference cell population has hepatotoxicity.
Conversely, when the reference cell population contains cells that have been
exposed
to a hepatotoxic agent, a similarity in expression pattern between the test
cell population and
the reference cell population indicates the test cell population has
hepatotoxicity. A difference
in expression between ACETA sequences in the test population and the reference
cell
population indicates the subject does not have hepatoxicity.
ACETA NUCLEIC ACIDS
Also provided in the invention are novel nucleic acid comprising a nucleic
acid
sequence selected from the group consisting of ACETA:1-10 and 43-54, or its
complement, as
well as vectors and cells including these nucleic acids.
Thus, one aspect of the invention pertains to isolated ACETA nucleic acid
molecules
that encode ACETA proteins or biologically active portions thereof. Also
included are nucleic
acid fragments sufficient for use as hybridization probes to identify ACETA-
encoding nucleic
acids (e.g., ACETA mRNA) and fragments for use as polymerase chain reaction
(PCR)
primers for the amplification or mutation of ACETA nucleic acid molecules. As
used herein,
the term "nucleic acid molecule" is intended to include DNA molecules (e.g.,
cDNA or
genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated
using
nucleotide analogs, and derivatives, fragments and homologs thereof. The
nucleic acid
molecule can be single-stranded or double-stranded, but preferably is double-
stranded DNA.
"Probes" refer to nucleic acid sequences of variable length, preferably
between at least
about 10 nucleotides (nt) or as many as about, e.g., 6,000 nt, depending on
use. Probes are
used in the detection of identical, similar, or complementary nucleic acid
sequences. Longer
length probes are usually obtained from a natural or recombinant source, are
highly specific
and much slower to hybridize than oligomers. Probes may be single- or double-
stranded and
designed to have specificity in PCR, membrane-based hybridization
technologies, or ELISA-
like technologies.
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An "isolated" nucleic acid molecule is one that is separated from other
nucleic acid
molecules which are present in the natural source of the nucleic acid.
Examples of isolated
nucleic acid molecules include, but are not limited to, recombinant DNA
molecules contained
in a vector, recombinant DNA molecules maintained in a heterologous host cell,
partially or
substantially purified nucleic acid molecules, and synthetic DNA or RNA
molecules.
Preferably, an "isolated" nucleic acid is free of sequences which naturally
flank the nucleic
acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in
the genomic DNA of
the organism from which the nucleic acid is derived. For example, in various
embodiments,
the isolated ACETA nucleic acid molecule can contain less than about SO kb, 25
kb, 5 kb, 4
kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally
flank the nucleic
acid molecule in genomic DNA of the cell from which the nucleic acid is
derived. Moreover,
an "isolated" nucleic acid molecule, such as a cDNA molecule, can be
substantially free of
other cellular material or culture medium when produced by recombinant
techniques, or of
chemical precursors or other chemicals when chemically synthesized.
A nucleic acid molecule of the present invention, e.g., a nucleic acid
molecule having
the nucleotide sequence of any of ACETA:l-10 and 43-54, or a complement of any
of these
nucleotide sequences, can be isolated using standard molecular biology
techniques and the
sequence information provided herein. Using all or a portion of these nucleic
acid sequences
as a hybridization probe, ACETA nucleic acid sequences can be isolated using
standard
hybridization and cloning techniques (e.g., as described in Sambrook et al.,
eds., MOLECULAR
CLONrnT~: A LABORAT'oRY MANUAL 2"d Ed., Cold Spring Harbor Laboratory Press,
Cold
Spring Harbor, NY, 1989; and Ausubel, et al., eds., CURRENT PROTOCOLS IN
MOLECULAR
BIOLOGY, John Wiley & Sons, New York, NY, 1993.)
A nucleic acid of the invention can be amplified using cDNA, mRNA or
alternatively,
genomic DNA, as a template and appropriate oligonucleotide primers according
to standard
PCR amplification techniques. The nucleic acid so amplified can be cloned into
an
appropriate vector and characterized by DNA sequence analysis. Furthermore,
oligonucleotides corresponding to ACETA nucleotide sequences can be prepared
by standard
synthetic techniques, e.g., using an automated DNA synthesizer.
As used herein, the term "oligonucleotide" refers to a series of linked
nucleotide
residues, which oligonucleotide has a sufficient number of nucleotide bases to
be used in a
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PCR reaction. A short oligonucleotide sequence may be based on, or designed
from, a
genomic or cDNA sequence and is used to amplify, confirm, or reveal the
presence of an
identical, similar or complementary DNA or RNA in a particular cell or tissue.
Oligonucleotides comprise portions of a nucleic acid sequence having at least
about 10 nt and
as many as 50 nt, preferably about 15 nt to 30 nt. They may be chemically
synthesized and
may be used as probes.
In another embodiment, an isolated nucleic acid molecule of the invention
comprises a
nucleic acid molecule that is a complement of the nucleotide sequence shown in
ACETA: 1-10
and 43-54 . In another embodiment, an isolated nucleic acid molecule of the
invention
comprises a nucleic acid molecule that is a complement of the nucleotide
sequence shown in
any of these sequences, or a portion of any of these nucleotide sequences. A
nucleic acid
molecule that is complementary to the nucleotide sequence shown in ACETA:l-10
and 43-54
is one that is sufficiently complementary to the nucleotide sequence shown,
such that it can
hydrogen bond with little or no mismatches to the nucleotide sequences shown,
thereby
forming a stable duplex.
As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen
base
pairing between nucleotides units of a nucleic acid molecule, and the term
"binding" means
the physical or chemical interaction between two polypeptides or compounds or
associated
polypeptides or compounds or combinations thereof. Binding includes ionic, non-
ionic, Von
der Waals, hydrophobic interactions, etc. A physical interaction can be either
direct or
indirect. Indirect interactions may be through or due to the effects of
another polypeptide or
compound. Direct binding refers to interactions that do not take place
through, or due to, the
effect of another polypeptide or compound, but instead are without other
substantial chemical
intermediates.
Moreover, the nucleic acid molecule of the invention can comprise only a
portion of
the nucleic acid sequence of ACETA:1-10 and 43-54 e.g., a fragment that can be
used as a
probe or primer or a fragment encoding a biologically active portion of ACETA.
Fragments
provided herein are defined as sequences of at least 6 (contiguous) nucleic
acids or at least 4
(contiguous) amino acids, a length sufficient to allow for specific
hybridization in the case of
nucleic acids or for specific recognition of an epitope in the case of amino
acids, respectively,
and are at most some portion less than a full length sequence. Fragments may
be derived from
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any contiguous portion of a nucleic acid or amino acid sequence of choice.
Derivatives are
nucleic acid sequences or amino acid sequences formed from the native
compounds either
directly or by modification or partial substitution. Analogs are nucleic acid
sequences or
amino acid sequences that have a structure similar to, but not identical to,
the native compound
but differs from it in respect to certain components or side chains. Analogs
may be synthetic
or from a different evolutionary origin and may have a similar or opposite
metabolic activity
compared to wild type.
Derivatives and analogs may be full length or other than full length, if the
derivative or
analog contains a modified nucleic acid or amino acid, as described below.
Derivatives or
analogs of the nucleic acids or proteins of the invention include, but are not
limited to,
molecules comprising regions that are substantially homologous to the nucleic
acids or
proteins of the invention, in various embodiments, by at least about 45%, 50%,
70%, 80%,
95%, 98%, or even 99% identity (with a preferred identity of 80-99%) over a
nucleic acid or
amino acid sequence of identical size or when compared to an aligned sequence
in which the
alignment is done by a computer homology program known in the art, or whose
encoding
nucleic acid is capable of hybridizing to the complement of a sequence
encoding the
aforementioned proteins under stringent, moderately stringent, or low
stringent conditions.
See e.g. Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &
Sons,
New York, NY, 1993, and below. An exemplary program is the Gap program
(Wisconsin
Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group,
University
Research Park, Madison, WI) using the default settings, which uses the
algorithm of Smith and
Waterman (Adv. Appl. Math., 1981, 2: 482-489, which in incorporated herein by
reference in
its entirety).
A "homologous nucleic acid sequence" or "homologous amino acid sequence," or
variations thereof, refer to sequences characterized by a homology at the
nucleotide level or
amino acid level as discussed above. Homologous nucleotide sequences encode
those
sequences coding for isoforms of a ACETA polypeptide. Isoforms can be
expressed in
different tissues of the same organism as a result of, for example,
alternative splicing of RNA.
Alternatively, isoforms can be encoded by different genes. In the present
invention,
homologous nucleotide sequences include nucleotide sequences encoding for a
ACETA
polypeptide of species other than humans, including, but not limited to,
mammals, and thus
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can include, e.g., mouse, rat, rabbit, dog, cat cow, horse, and other
organisms. Homologous
nucleotide sequences also include, but are not limited to, naturally occurring
allelic variations
and mutations of the nucleotide sequences set forth herein. A homologous
nucleotide
sequence does not, however, include the nucleotide sequence encoding a human
ACETA
protein. Homologous nucleic acid sequences include those nucleic acid
sequences that encode
conservative amino acid substitutions (see below) in a ACETA polypeptide, as
well as a
polypeptide having a ACETA activity. A homologous amino acid sequence does not
encode
the amino acid sequence of a human ACETA polypeptide.
The nucleotide sequence determined from the cloning of human ACETA genes
allows
for the generation of probes and primers designed for use in identifying
and/or cloning
ACETA homologues in other cell types, e.g., from other tissues, as well as
ACETA
homologues from other mammals. The probe/primer typically comprises a
substantially
purified oligonucleotide. The oligonucleotide typically comprises a region of
nucleotide
sequence that hybridizes under stringent conditions to at least about 12, 25,
50, 100, 150, 200,
250, 300, 350 or 400 consecutive sense strand nucleotide sequence of a nucleic
acid
comprising a ACETA sequence, or an anti-sense strand nucleotide sequence of a
nucleic acid
comprising a ACETA sequence, or of a naturally occurring mutant of these
sequences.
Probes based on human ACETA nucleotide sequences can be used to detect
transcripts
or genomic sequences encoding the same or homologous proteins. In various
embodiments,
the probe further comprises a label group attached thereto, e.g., the label
group can be a
radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such
probes can be
used as a part of a diagnostic test kit for identifying cells or tissue which
misexpress a ACETA
protein, such as by measuring a level of a ACETA-encoding nucleic acid in a
sample of cells
from a subject e.g., detecting ACETA mRNA levels or determining whether a
genomic
ACETA gene has been mutated or deleted.
"A polypeptide having a biologically active portion of ACETA" refers to
polypeptides
exhibiting activity similar, but not necessarily identical to, an activity of
a polypeptide of the
present invention, including mature forms, as measured in a particular
biological assay, with or
without dose dependency. A nucleic acid fragment encoding a "biologically
active portion of
ACETA" can be prepared by isolating a portion of ACETA:l-10 and 43-54, that
encodes a
polypeptide having a ACETA biological activity, expressing the encoded portion
of ACETA
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protein (e.g., by recombinant expression in vitro) and assessing the activity
of the encoded
portion of ACETA. For example, a nucleic acid fragment encoding a biologically
active
portion of a ACETA polypeptide can optionally include an ATP-binding domain.
In another
embodiment, a nucleic acid fragment encoding a biologically active portion of
ACETA
includes one or more regions.
ACETA VARIANTS
The invention further encompasses nucleic acid molecules that differ from the
disclosed or referenced ACETA nucleotide sequences due to degeneracy of the
genetic code.
These nucleic acids thus encode the same ACETA protein as that encoded by
nucleotide
sequence comprising a ACETA nucleic acid as shown in, e.g., ACETA:l-10 and 43-
54
In addition to the rat ACETA nucleotide sequence shown in ACETA:1-10 and 43-
54, it
will be appreciated by those skilled in the art that DNA sequence
polymorphisms that lead to
changes in the amino acid sequences of a ACETA polypeptide may exist within a
population
(e.g., the human population). Such genetic polymorphism in the ACETA gene may
exist
among individuals within a population due to natural allelic variation. As
used herein, the
terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising
an open
reading frame encoding a ACETA protein, preferably a mammalian ACETA protein.
Such
natural allelic variations can typically result in 1-5% variance in the
nucleotide sequence of the
ACETA gene. Any and all such nucleotide variations and resulting amino acid
polymorphisms in ACETA that are the result of natural allelic variation and
that do not alter
the functional activity of ACETA are intended to be within the scope of the
invention.
Moreover, nucleic acid molecules encoding ACETA proteins from other species,
and
thus that have a nucleotide sequence that differs from the human sequence of
ACETA:l-10
and 43-54, are intended to be within the scope of the invention. Nucleic acid
molecules
corresponding to natural allelic variants and homologues of the ACETA DNAs of
the
invention can be isolated based on their homology to the human ACETA nucleic
acids
disclosed herein using the human cDNAs, or a portion thereof, as a
hybridization probe
according to standard hybridization techniques under stringent hybridization
conditions. For
example, a soluble human ACETA DNA can be isolated based on its homology to
human
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membrane-bound ACETA. Likewise, a membrane-bound human ACETA DNA can be
isolated based on its homology to soluble human ACETA.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the
invention is at least 6 nucleotides in length and hybridizes under stringent
conditions to the
nucleic acid molecule comprising the nucleotide sequence of ACETA:1-10 and 43-
54. In
another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250 or 500
nucleotides in
length. In another embodiment, an isolated nucleic acid molecule of the
invention hybridizes
to the coding region. As used herein, the term "hybridizes under stringent
conditions" is
intended to describe conditions for hybridization and washing under which
nucleotide
sequences at least 60% homologous to each other typically remain hybridized to
each other.
Homologs (i.e., nucleic acids encoding ACETA proteins derived from species
other
than human) or other related sequences (e.g., paralogs) can be obtained by
low, moderate or
high stringency hybridization with all or a portion of the particular human
sequence as a probe
using methods well known in the art for nucleic acid hybridization and
cloning.
1 S As used herein, the phrase "stringent hybridization conditions" refers to
conditions
under which a probe, primer or oligonucleotide will hybridize to its target
sequence, but to no
other sequences. Stringent conditions are sequence-dependent and will be
different in different
circumstances. Longer sequences hybridize specifically at higher temperatures
than shorter
sequences. Generally, stringent conditions are selected to be about 5°C
lower than the thermal
melting point (Tm) for the specific sequence at a defined ionic strength and
pH. The Tm is the
temperature (under defined ionic strength, pH and nucleic acid concentration)
at which 50% of
the probes complementary to the target sequence hybridize to the target
sequence at
equilibrium. Since the target sequences are generally present at excess, at
Tm, 50% of the
probes are occupied at equilibrium. Typically, stringent conditions will be
those in which the
salt concentration is less than about 1.0 M sodium ion, typically about 0.01
to 1.0 M sodium
ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about
30°C for short probes,
primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about
60°C for longer probes,
primers and oligonucleotides. Stringent conditions may also be achieved with
the addition of
destabilizing agents, such as formamide.
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Stringent conditions are known to those skilled in the art and can be found in
CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
Preferably, the conditions are such that sequences at least about 65%, 70%,
75%, 85%, 90%,
95%, 98%, or 99% homologous to each other typically remain hybridized to each
other.
A non-limiting example of stringent hybridization conditions is hybridization
in a high salt
buffer comprising 6X SSC, 50 mM Tris-HC1 (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02%
Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C.
This hybridization
is followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C. An
isolated nucleic
acid molecule of the invention that hybridizes under stringent conditions to
the sequence of
ACETA:1-10 and 43-54 corresponds to a naturally occurring nucleic acid
molecule. As used
herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA
molecule
having a nucleotide sequence that occurs in nature (e.g., encodes a natural
protein).
In a second embodiment, a nucleic acid sequence that is hybridizable to the
nucleic
acid molecule comprising the nucleotide sequence of ACETA:l-10 and 43-54 or
fragments,
analogs or derivatives thereof, under conditions of moderate stringency is
provided. A
non-limiting example of moderate stringency hybridization conditions are
hybridization in 6X
SSC, SX Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA
at
55°C, followed by one or more washes in 1X SSC, 0.1% SDS at
37°C. Other conditions of
moderate stringency that may be used are well known in the art. See, e.g.,
Ausubel et al.
(eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY,
and
Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, StOCkton
Press,
NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid
molecule
comprising the nucleotide sequence of ACETA:1-10 and 43-54or fragments,
analogs or
derivatives thereof, under conditions of low stringency, is provided. A non-
limiting example
of low stringency hybridization conditions are hybridization in 35% formamide,
SX SSC, 50
mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml
denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C,
followed by one or more
washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at
50°C. Other
conditions of low stringency that may be used are well known in the art (e.g.,
as employed for
cross-species hybridizations). See, e.g., Ausubel et al. (eds.), 1993, CURRENT
PROTOCOLS IN
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MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER
AND
EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo et al., 1981, Proc
Natl
Acad Sci USA 78: 6789-6792.
CONSERVATIVE MUTATIONS
In addition to naturally-occurring allelic variants of the ACETA sequence that
may
exist in the population, the skilled artisan will further appreciate that
changes can be
introduced into an ACETA nucleic acid or directly into an ACETA polypeptide
sequence
without altering the functional ability of the ACETA protein. In some
embodiments, the
nucleotide sequence of ACETA:1-10 and 43-54will be altered, thereby leading to
changes in
the amino acid sequence of the encoded ACETA protein. For example, nucleotide
substitutions that result in amino acid substitutions at various "non-
essential" amino acid
residues can be made in the sequence of ACETA:1-10 and 43-54. A "non-
essential" amino
acid residue is a residue that can be altered from the wild-type sequence of
ACETA without
altering the biological activity, whereas an "essential" amino acid residue is
required for
biological activity. For example, amino acid residues that are conserved among
the ACETA
proteins of the present invention, are predicted to be particularly unamenable
to alteration.
In addition, amino acid residues that are conserved among family members of
the
ACETA proteins of the present invention, are also predicted to be particularly
unamenable to
alteration. As such, these conserved domains are not likely to be amenable to
mutation. Other
amino acid residues, however, (e.g., those that are not conserved or only semi-
conserved
among members of the ACETA proteins) may not be essential for activity and
thus are likely
to be amenable to alteration.
Another aspect of the invention pertains to nucleic acid molecules encoding
ACETA
proteins that contain changes in amino acid residues that are not essential
for activity. Such
ACETA proteins differ in amino acid sequence from the amino acid sequences of
polypeptides
encoded by nucleic acids containing ACETA:1-10 and 43-54, yet retain
biological activity. In
one embodiment, the isolated nucleic acid molecule comprises a nucleotide
sequence encoding
a protein, wherein the protein comprises an amino acid sequence at least about
45%
homologous, more preferably 60%, and still more preferably at least about 70%,
80%, 90%,
95%, 98%, and most preferably at least about 99% homologous to the amino acid
sequence of
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the amino acid sequences of polypeptides encoded by nucleic acids comprising
ACETA:1-10
and 43-54.
An isolated nucleic acid molecule encoding a ACETA protein homologous to can
be
created by introducing one or more nucleotide substitutions, additions or
deletions into the
nucleotide sequence of a nucleic acid comprising ACETA:1-10 and 43-54, such
that one or
more amino acid substitutions, additions or deletions are introduced into the
encoded protein.
Mutations can be introduced into a nucleic acid comprising ACETA:1-10 and 43-
54 by
standard techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis.
Preferably, conservative amino acid substitutions are made at one or more
predicted
non-essential amino acid residues. A "conservative amino acid substitution" is
one in which
the amino acid residue is replaced with an amino acid residue having a similar
side chain.
Families of amino acid residues having similar side chains have been defined
in the art. These
families include amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic
side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains
(e.g., glycine,
asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g.,
alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan),
beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic
side chains (e.g.,
tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted
nonessential amino acid
residue in ACETA is replaced with another amino acid residue from the same
side chain
family. Alternatively, in another embodiment, mutations can be introduced
randomly along all
or part of a ACETA coding sequence, such as by saturation mutagenesis, and the
resultant
mutants can be screened for ACETA biological activity to identify mutants that
retain activity.
Following mutagenesis of the nucleic acids the encoded protein can be
expressed by any
recombinant technology known in the art and the activity of the protein can be
determined.
2~ In one embodiment, a mutant ACETA protein can be assayed for ( 1 ) the
ability to form
protein:protein interactions with other ACETA proteins, other cell-surface
proteins, or
biologically active portions thereof, (2) complex formation between a mutant
ACETA protein
and a ACETA ligand; (3) the ability of a mutant ACETA protein to bind to an
intracellular
target protein or biologically active portion thereof; (e.g., avidin
proteins); (4) the ability to
bind ATP; or (5) the ability to specifically bind a ACETA protein antibody.
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In other specific embodiments, the nucleic acid is RNA or DNA.
ANTI SENSE
Another aspect of the invention pertains to isolated antisense nucleic acid
molecules
that are hybridizable to or complementary to the nucleic acid molecule
comprising the
nucleotide sequence of a ACETA sequence or fragments, analogs or derivatives
thereof. An
"antisense" nucleic acid comprises a nucleotide sequence that is complementary
to a "sense"
nucleic acid encoding a protein, e.g., complementary to the coding strand of a
double-stranded
cDNA molecule or complementary to an mRNA sequence. In specific aspects,
antisense
nucleic acid molecules are provided that comprise a sequence complementary to
at least about
10, 25, S0, 100, 250 or 500 nucleotides or an entire ACETA coding strand, or
to only a portion
thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and
analogs of a
ACETA protein, or antisense nucleic acids complementary to a nucleic acid
comprising a
ACETA nucleic acid sequence are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a
"coding
region" of the coding strand of a nucleotide sequence encoding ACETA. The term
"coding
region" refers to the region of the nucleotide sequence comprising codons
which are translated
into amino acid residues. In another embodiment, the antisense nucleic acid
molecule is
antisense to a "noncoding region" of the coding strand of a nucleotide
sequence encoding
ACETA. The term "noncoding region" refers to 5' and 3' sequences which flank
the coding
region that are not translated into amino acids (i. e., also referred to as 5'
and 3' untranslated
regions).
Given the coding strand sequences encoding ACETA disclosed herein, antisense
nucleic acids of the invention can be designed according to the rules of
Watson and Crick or
Hoogsteen base pairing. The antisense nucleic acid molecule can be
complementary to the
entire coding region of ACETA mRNA, but more preferably is an oligonucleotide
that is
antisense to only a portion of the coding or noncoding region of ACETA mRNA.
For
example, the antisense oligonucleotide can be complementary to the region
surrounding the
translation start site of ACETA mRNA. An antisense oligonucleotide can be, for
example,
about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An
antisense nucleic acid of
the invention can be constructed using chemical synthesis or enzymatic
ligation reactions
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using procedures known in the art. For example, an antisense nucleic acid
(e.g., an antisense
oligonucleotide) can be chemically synthesized using naturally occurring
nucleotides or
variously modified nucleotides designed to increase the biological stability
of the molecules or
to increase the physical stability of the duplex formed between the antisense
and sense nucleic
acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides
can be used.
Examples of modified nucleotides that can be used to generate the antisense
nucleic
acid include: 5-fluorouracil, 5-bromouracil, ~-chlorouracil, 5-iodouracil,
hypoxanthine,
xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-
carboxymethylaminomethyl-
2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-
galactosylqueosine,
inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-
dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-
adenine,
7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine,
pseudouracil,
1 S queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-
thiouracil, 5-methyluracil,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-
thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
Alternatively, the
antisense nucleic acid can be produced biologically using an expression vector
into which a
nucleic acid has been subcloned in an antisense orientation (i. e., RNA
transcribed from the
inserted nucleic acid will be of an antisense orientation to a target nucleic
acid of interest,
described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically
administered to a
subject or generated isz situ such that they hybridize with or bind to
cellular mRNA and/or
genomic DNA encoding a ACETA protein to thereby inhibit expression of the
protein, e.g., by
inhibiting transcription and/or translation. The hybridization can be by
conventional
nucleotide complementarity to form a stable duplex, or, for example, in the
case of an
antisense nucleic acid molecule that binds to DNA duplexes, through specific
interactions in
the major groove of the double helix. An example of a route of administration
of antisense
nucleic acid molecules of the invention includes direct injection at a tissue
site. Alternatively,
antisense nucleic acid molecules can be modified to target selected cells and
then administered
systemically. For example, for systemic administration, antisense molecules
can be modified
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such that they specifically bind to receptors or antigens expressed on a
selected cell surface,
e.g., by linking the antisense nucleic acid molecules to peptides or
antibodies that bind to cell
surface receptors or antigens. The antisense nucleic acid molecules can also
be delivered to
cells using the vectors described herein. To achieve sufficient intracellular
concentrations of
antisense molecules, vector constructs in which the antisense nucleic acid
molecule is placed
under the control of a strong pol II or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the
invention is an
a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms
specific
double-stranded hybrids with complementary RNA in which, contrary to the usual
(3-units, the
strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res
15: 6625-6641). The
antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide
(moue et cal.
(1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA -DNA analogue (moue
et al.
(1987) FEBS Lett 215: 327-330).
RIBOZYMES AND PNA MOIETIES
In still another embodiment, an antisense nucleic acid of the invention is a
ribozyme.
Ribozymes are catalytic RNA molecules with ribonuclease activity that are
capable of cleaving
a single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region.
Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and
Gerlach (1988)
Nature 334:585-591)) can be used to catalytically cleave ACETA mRNA
transcripts to thereby
inhibit translation of ACETA mRNA. A ribozyme having specificity for a ACETA-
encoding
nucleic acid can be designed based upon the nucleotide sequence of a ACETA DNA
disclosed
herein. For example, a derivative of a Tetrahymena L-19 IVS RNA can be
constructed in
which the nucleotide sequence of the active site is complementary to the
nucleotide sequence
to be cleaved in a ACETA-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No.
4,987,071;
and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, ACETA mRNA can be used
to select a
catalytic RNA having a specific ribonuclease activity from a pool of RNA
molecules. See,
e.g., Bartel et al., (1993) Science 261:1411-1418.
Alternatively, ACETA gene expression can be inhibited by targeting nucleotide
sequences complementary to the regulatory region of a ACETA nucleic acid
(e.g., the
ACETA promoter andlor enhancers) to form triple helical structures that
prevent transcription
CA 02377768 2001-12-18
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of the ACETA gene in target cells. See generally, Helene. (1991) Anticancer
Drug Des. 6:
569-84; Helene. et al. (1992) Ann. N. Y. Acad. Sci. 660:27-36; and Maher
(1992) Bioassays 14:
807-15.
In various embodiments, the nucleic acids of ACETA can be modified at the base
moiety, sugar moiety or phosphate backbone to improve, e.g., the stability,
hybridization, or
solubility of the molecule. For example, the deoxyribose phosphate backbone of
the nucleic
acids can be modified to generate peptide nucleic acids (see Hyrup et al.
(1996) Bioorg Med
Chenz 4: 5-23). As used herein, the terms "peptide nucleic acids" or "PNAs"
refer to nucleic
acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is
replaced by
a pseudopeptide backbone and only the four natural nucleobases are retained.
The neutral
backbone of PNAs has been shown to allow for specific hybridization to DNA and
RNA under
conditions of low ionic strength. The synthesis of PNA oligomers can be
performed using
standard solid phase peptide synthesis protocols as described in Hyrup et al.
(1996) above;
Pent'-O'Keefe et al. (1996) PNAS 93: 14670-675.
PNAs of ACETA can be used in therapeutic and diagnostic applications. For
example,
PNAs can be used as antisense or antigene agents for sequence-specific
modulation of gene
expression by, e.g., inducing transcription or translation arrest or
inhibiting replication. PNAs
of ACETA can also be used, e.g., in the analysis of single base pair mutations
in a gene by,
e.g., PNA directed PCR clamping; as artificial restriction enzymes when used
in combination
with other enzymes, e.g., S 1 nucleases (Hyrup B. (1996) above); or as probes
or primers for
DNA sequence and hybridization (Hyrup et al. (1996), above; Pent'-O'Keefe
(1996), above).
In another embodiment, PNAs of ACETA can be modified, e.g., to enhance their
stability or cellular uptake, by attaching lipophilic or other helper groups
to PNA, by the
formation of PNA-DNA chimeras, or by the use of liposomes or other techniques
of drug
delivery known in the art. For example, PNA-DNA chimeras of ACETA can be
generated that
may combine the advantageous properties of PNA and DNA. Such chimeras allow
DNA
recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the
DNA portion
while the PNA portion would provide high binding affinity and specificity. PNA-
DNA
chimeras can be linked using linkers of appropriate lengths selected in teens
of base stacking,
number of bonds between the nucleobases, and orientation (Hyrup ( 1996)
above). The
synthesis of PNA-DNA chimeras can be perfornzed as described in Hyrup (1996)
above and
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Finn et al. (1996) Nucl Acids Res 24: 3357-63. For example, a DNA chain can be
synthesized
on a solid support using standard phosphoramidite coupling chemistry, and
modified
nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine
phosphoramidite, can
be used between the PNA and the 5' end of DNA (Mag et al. (1989) Na~cl Acid
Res 17:
5973-88). PNA monomers are then coupled in a stepwise manner to produce a
chimeric
molecule with a 5' PNA segment and a 3' DNA segment (Finn et al. (1996)
above).
Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and
a 3' PNA
segment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-11124.
In other embodiments, the oligonucleotide may include other appended groups
such as
peptides (e.g., for targeting host cell receptors in vivo), or agents
facilitating transport across
the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci.
U.S.A.
86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT
Publication No.
W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No.
W089/10134). In
addition, oligonucleotides can be modified with hybridization triggered
cleavage agents (See,
e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents.
(See, e.g., Zon, 1988,
Pharm. Res. 5: 539-549). To this end, the oligonucleotide may be conjugated to
another
molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a
transport agent, a
hybridization-triggered cleavage agent, etc.
ACETA POLYPEPTIDES
One aspect of the invention pertains to isolated ACETA proteins, and
biologically
active portions thereof, or derivatives, fragments, analogs or homologs
thereof. Also provided
are polypeptide fragments suitable for use as immunogens to raise anti-ACETA
antibodies. In
one embodiment, native ACETA proteins can be isolated from cells or tissue
sources by an
appropriate purification scheme using standard protein purification
techniques. In another
embodiment, ACETA proteins are produced by recombinant DNA techniques.
Alternative to
recombinant expression, a ACETA protein or polypeptide can be synthesized
chemically using
standard peptide synthesis techniques.
An "isolated" or "purified" protein or biologically active portion thereof is
substantially
free of cellular material or other contaminating proteins from the cell or
tissue source from
which the ACETA protein is derived, or substantially free from chemical
precursors or other
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chemicals when chemically synthesized. The language "substantially free of
cellular material"
includes preparations of ACETA protein in which the protein is separated from
cellular
components of the cells from which it is isolated or recombinantly produced.
In one
embodiment, the language "substantially free of cellular material" includes
preparations of
ACETA protein having less than about 30% (by dry weight) of non-ACETA protein
(also
referred to herein as a "contaminating protein"), more preferably less than
about 20% of
non-ACETA protein, still more preferably less than about 10% of non-ACETA
protein, and
most preferably less than about 5% non-ACETA protein. When the ACETA protein
or
biologically active portion thereof is recombinantly produced, it is also
preferably substantially
free of culture medium, i. e., culture medium represents less than about 20%,
more preferably
less than about 10%, and most preferably less than about 5% of the volume of
the protein
preparation.
The language "substantially free of chemical precursors or other chemicals"
includes
preparations of ACETA protein in which the protein is separated from chemical
precursors or
other chemicals that are involved in the synthesis of the protein. In one
embodiment, the
language "substantially free of chemical precursors or other chemicals"
includes preparations
of ACETA protein having less than about 30% (by dry weight) of chemical
precursors or
non-ACETA chemicals, more preferably less than about 20% chemical precursors
or
non-ACETA chemicals, still more preferably less than about 10% chemical
precursors or
non-ACETA chemicals, and most preferably less than about S% chemical
precursors or
non-ACETA chemicals.
Biologically active portions of a ACETA protein include peptides comprising
amino
acid sequences sufficiently homologous to or derived from the amino acid
sequence of the
ACETA protein, e.g., the amino acid sequence encoded by a nucleic acid
comprising ACETA
1-10 and 43-54 that include fewer amino acids than the full length ACETA
proteins, and
exhibit at least one activity of a ACETA protein. Typically, biologically
active portions
comprise a domain or motif with at least one activity of the ACETA protein. A
biologically
active portion of a ACETA protein can be a polypeptide which is, for example,
10, 25, S0, 100
or more amino acids in length.
A biologically active portion of a ACETA protein of the present invention may
contain
at least one of the above-identified domains conserved between the ACETA
proteins. An
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alternative biologically active portion of a ACETA protein may contain at
least two of the
above-identified domains. Another biologically active portion of a ACETA
protein may
contain at least three of the above-identified domains. Yet another
biologically active portion
of a ACETA protein of the present invention may contain at least four of the
above-identified
domains.
Moreover, other biologically active portions, in which other regions of the
protein are
deleted, can be prepared by recombinant techniques and evaluated for one or
more of the
functional activities of a native ACETA protein.
In some embodiments, the ACETA protein is substantially homologous to one of
these
ACETA proteins and retains its the functional activity, yet differs in amino
acid sequence due
to natural allelic variation or mutagenesis, as described in detail below.
In specific embodiments, the invention includes an isolated polypeptide
comprising an
amino acid sequence that is 80% or more identical to the sequence of a
polypeptide whose
expression is modulated in a mammal to which hepatotoxic agent is
administered.
1 S DETERMINING HOMOLOGY BETWEEN TWO OR nlORE SEQUENCES
To determine the percent homology of two amino acid sequences or of two
nucleic
acids, the sequences are aligned for optimal comparison purposes (e.g., gaps
can be introduced
in the sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a
second amino or nucleic acid sequence). The amino acid residues or nucleotides
at
corresponding amino acid positions or nucleotide positions are then compared.
When a
position in the first sequence is occupied by the same amino acid residue or
nucleotide as the
corresponding position in the second sequence, then the molecules are
homologous at that
position (i.e., as used herein amino acid or nucleic acid "homology" is
equivalent to amino
acid or nucleic acid "identity").
The nucleic acid sequence homology may be determined as the degree of identity
between two sequences. The homology may be determined using computer programs
known
in the art, such as GAP software provided in the GCG program package. See
Needlemari and
Wunsch 1970 JMoI Biol 48: 443-453. Using GCG GAP software with the following
settings
for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP
extension penalty
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of 0.3, the coding region of the analogous nucleic acid sequences referred to
above exhibits a
degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%,
or 99%, with
the CDS (encoding) part of a DNA sequence comprising ACETA: 1-10 and 43-54..
The term "sequence identity" refers to the degree to which two polynucleotide
or
polypeptide sequences are identical on a residue-by-residue basis over a
particular region of
comparison. The term "percentage of sequence identity" is calculated by
comparing two
optimally aligned sequences over that region of comparison, determining the
number of
positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I,
in the case of
nucleic acids) occurs in both sequences to yield the number of matched
positions, dividing the
number of matched positions by the total number of positions in the region of
comparison (i.e.,
the window size), and multiplying the result by 100 to yield the percentage of
sequence
identity. The term "substantial identity" as used herein denotes a
characteristic of a
polynucleotide sequence, wherein the polynucleotide comprises a sequence that
has at least 80
percent sequence identity, preferably at least 85 percent identity and often
90 to 95 percent
sequence identity, more usually at least 99 percent sequence identity as
compared to a
reference sequence over a comparison region.
CHIMERIC AND FUSION PROTEINS
The invention also provides ACETA chimeric or fusion proteins. As used herein,
an
ACETA "chimeric protein" or "fusion protein" comprises an ACETA polypeptide
operatively
linked to a non-ACETA polypeptide. A "ACETA polypeptide" refers to a
polypeptide having
an amino acid sequence corresponding to ACETA, whereas a "non-ACETA
polypeptide"
refers to a polypeptide having an amino acid sequence corresponding to a
protein that is not
substantially homologous to the ACETA protein, e.g., a protein that is
different from the
ACETA protein and that is derived from the same or a different organism.
Within an ACETA
fusion protein the ACETA polypeptide can correspond to all or a portion of an
ACETA
protein. In one embodiment, an ACETA fusion protein comprises at least one
biologically
active portion of an ACETA protein. In another embodiment, an ACETA fusion
protein
comprises at least two biologically active portions of an ACETA protein. In
yet another
embodiment, an ACETA fusion protein comprises at least three biologically
active portions of
an ACETA protein. Within the fusion protein, the term "operatively linked" is
intended to
CA 02377768 2001-12-18
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indicate that the ACETA polypeptide and the non-ACETA polypeptide are fused in-
frame to
each other. The non-ACETA polypeptide can be fused to the N-terminus or C-
terminus of the
ACETA polypeptide.
For example, in one embodiment an ACETA fusion protein comprises an ACETA
domain operably linked to the extracellular domain of a second protein. Such
fusion proteins
can be further utilized in screening assays for compounds which modulate ACETA
activity
(such assays are described in detail below).
In yet another embodiment, the fusion protein is a GST-ACETA fusion protein in
which the ACETA sequences are fused to the C-terminus of the GST (i.e.,
glutathione
S-transferase) sequences. Such fusion proteins can facilitate the purification
of recombinant
ACETA.
In another embodiment, the fusion protein is an ACETA protein containing a
heterologous signal sequence at its N-terminus. For example, a native ACETA
signal
sequence can be removed and replaced with a signal sequence from another
protein. In certain
host cells (e.g:, mammalian host cells), expression and/or secretion of ACETA
can be
increased through use of a heterologous signal sequence.
In yet another embodiment, the fusion protein is an ACETA-immunoglobulin
fusion
protein in which the ACETA sequences comprising one or more domains are fused
to
sequences derived from a member of the immunoglobulin protein family. The
ACETA-immunoglobulin fusion proteins of the invention can be incorporated into
pharmaceutical compositions and administered to a subject to inhibit an
interaction between a
ACETA ligand and a ACETA protein on the surface of a cell, to thereby suppress
ACETA-mediated signal transduction iJZ vivo. The ACETA-immunoglobulin fusion
proteins
can be used to affect the bioavailability of an ACETA cognate ligand.
Inhibition of the
ACETA ligand/ACETA interaction may be useful therapeutically for both the
treatments of
proliferative and differentiative disorders, as well as modulating (e.g.
promoting or inhibiting)
cell survival. Moreover, the ACETA-immunoglobulin fusion proteins of the
invention can be
used as immunogens to produce anti-ACETA antibodies in a subject, to purify
ACETA
ligands, and in screening assays to identify molecules that inhibit the
interaction of ACETA
with a ACETA ligand.
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An ACETA chimeric or fusion protein of the invention can be produced by
standard
recombinant DNA techniques. For example, DNA fragments coding for the
different
polypeptide sequences are ligated together in-frame in accordance with
conventional
techniques, e.g., by employing blunt-ended or stagger-ended termini for
ligation, restriction
enzyme digestion to provide for appropriate termini, filling-in of cohesive
ends as appropriate,
alkaline phosphatase treatment to avoid undesirable joining, and enzymatic
ligation. In
another embodiment, the fusion gene can be synthesized by conventional
techniques including
automated DNA synthesizers. Alternatively, PCR amplification of gene fragments
can be
carned out using anchor primers that give rise to complementary overhangs
between two
consecutive gene fragments that can subsequently be annealed and reamplified
to generate a
chimeric gene sequence (see, for example, Ausubel et al. (eds.) CURRENT
PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors
are
commercially available that already encode a fusion moiety (e.g., a GST
polypeptide). An
ACETA-encoding nucleic acid can be cloned into such an expression vector such
that the
fusion moiety is linked in-frame to the ACETA protein.
ACETA AGONISTS AND ANTAGONISTS
The present invention also pertains to variants of the ACETA proteins that
function as
either ACETA agonists (mimetics) or as ACETA antagonists. Variants of the
ACETA protein
can be generated by mutagenesis, e.g., discrete point mutation or truncation
of the ACETA
protein. An agonist of the ACETA protein can retain substantially the same, or
a subset of, the
biological activities of the naturally occurring form of the ACETA protein. An
antagonist of
the ACETA protein can inhibit one or more of the activities of the naturally
occurring form of
the ACETA protein by, for example, competitively binding to a downstream or
upstream
member of a cellular signaling cascade which includes the ACETA protein. Thus,
specific
biological effects can be elicited by treatment with a variant of limited
function. In one
embodiment, treatment of a subject with a variant having a subset of the
biological activities of
the naturally occurring form of the protein has fewer side effects in a
subject relative to
treatment with the naturally occurring form of the ACETA proteins.
Variants of the ACETA protein that function as either ACETA agonists
(mimetics) or
as ACETA antagonists can be identified by screening combinatorial libraries of
mutants, e.g.,
47
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truncation mutants, of the ACETA protein for ACETA protein agonist or
antagonist activity.
In one embodiment, a variegated library of ACETA variants is generated by
combinatorial
mutagenesis at the nucleic acid level and is encoded by a variegated gene
library. A variegated
library of ACETA variants can be produced by, for example, enzymatically
ligating a mixture
of synthetic oligonucleotides into gene sequences such that a degenerate set
of potential
ACETA sequences is expressible as individual polypeptides, or alternatively,
as a set of larger
fusion proteins (e.g., for phage display) containing the set of ACETA
sequences therein.
There are a variety of methods which can be used to produce libraries of
potential ACETA
variants from a degenerate oligonucleotide sequence. Chemical synthesis of a
degenerate gene
I 0 sequence can be performed in an automatic DNA synthesizer, and the
synthetic gene then
ligated into an appropriate expression vector. Use of a degenerate set of
genes allows for the
provision, in one mixture, of all of the sequences encoding the desired set of
potential ACETA
sequences. Methods for synthesizing degenerate oligonucleotides are known in
the art (see,
e.g., Narang (1983) Tetrc~l2edron 39:3; Itakura et al. (1984) Annu Rev
Biochena 53:323; Itakura
et al. (1984) Science 198:1056; Ike et al. (1983) Nucl Acid Res 11:477.
POLYPEPTIDE LIBRARIES
In addition, libraries of fragments of the ACETA protein coding sequence can
be used
to generate a variegated population of ACETA fragments for screening and
subsequent
selection of variants of an ACETA protein. In one embodiment, a library of
coding sequence
fragments can be generated by treating a double stranded PCR fragment of a
ACETA coding
sequence with a nuclease under conditions wherein nicking occurs only about
once per
molecule, denaturing the double stranded DNA, renaturing the DNA to form
double stranded
DNA that can include sense/antisense pairs from different nicked products,
removing single
stranded portions from reformed duplexes by treatment with S 1 nuclease, and
ligating the
resulting fragment library into an expression vector. By this method, an
expression library can
be derived which encodes N-terminal and internal fragments of various sizes of
the ACETA
protein.
Several techniques are known in the art for screening gene products of
combinatorial
libraries made by point mutations or truncation, and for screening cDNA
libraries for gene
products having a selected property. Such techniques are adaptable for rapid
screening of the
48
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gene libraries generated by the combinatorial mutagenesis of ACETA proteins.
The most
widely used techniques, which are amenable to high throughput analysis, for
screening large
gene libraries typically include cloning the gene library into replicable
expression vectors,
transforming appropriate cells with the resulting library of vectors, and
expressing the
combinatorial genes under conditions in which detection of a desired activity
facilitates
isolation of the vector encoding the gene whose product was detected.
Recursive ensemble
mutagenesis (REM), a new technique that enhances the frequency of functional
mutants in the
libraries, can be used in combination with the screening assays to identify
ACETA variants
(Arkin and Yourvan (1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein
Engineering
6:327-331).
ANTI-ACETA ANTIBODIES
An isolated ACETA protein, or a portion or fragment thereof, can be used as an
immunogen to generate antibodies that bind ACETA using standard techniques for
polyclonal
and monoclonal antibody preparation. The full-length ACETA protein can be used
or,
alternatively, the invention provides antigenic peptide fragments of ACETA for
use as
immunogens. The antigenic peptide of ACETA comprises at least 8 amino acid
residues of
the amino acid sequence encoded by a nucleic acid comprising the nucleic acid
sequence
shown in ACETA:1-10 and 43-54 and encompasses an epitope of ACETA such that an
antibody raised against the peptide forms a specific immune complex with
ACETA.
Preferably, the antigenic peptide comprises at least 10 amino acid residues,
more preferably at
least 15 amino acid residues, even more preferably at least 20 amino acid
residues, and most
preferably at least 30 amino acid residues. Preferred epitopes encompassed by
the antigenic
peptide are regions of ACETA that are located on the surface of the protein,
e.g., hydrophilic
regions. As a means for targeting antibody production, hydropathy plots
showing regions of
hydrophilicity and hydrophobicity may be generated by any method well known in
the art,
including, for example, the Kyte Doolittle or the Hopp Woods methods, either
with or without
Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci.
USA 78:
3824-3828; Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each
incorporated herein by
reference in their entirety.
ACETA polypeptides or derivatives, fragments, analogs or homologs thereof, may
be
utilized as immunogens in the generation of antibodies that immunospecifically-
bind these
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protein components. The term "antibody" as used herein refers to
immunoglobulin molecules
and immunologically active portions of immunoglobulin molecules, i.e.,
molecules that
contain an antigen binding site that specifically binds (immunoreacts with) an
antigen. Such
antibodies include, but are not limited to, polyclonal, monoclonal, chimeric,
single chain, Fw
and F~ab.~~ fragments, and an Fay expression library. Various procedures known
within the art
may be used for the production of polyclonal or monoclonal antibodies to an
ACETA protein
sequence, or derivatives, fragments, analogs or homologs thereof. Some of
these proteins are
discussed below.
For the production of polyclonal antibodies, various suitable host animals
(e.g., rabbit,
goat, mouse or other mammal) may be immunized by injection with the native
protein, or a
synthetic variant thereof, or a derivative of the foregoing. An appropriate
immunogenic
preparation can contain, for example, recombinantly expressed ACETA protein or
a
chemically synthesized ACETA polypeptide. The preparation can further include
an adjuvant.
Various adjuvants used to increase the immunological response include, but are
not limited to,
Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide),
surface active
substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions,
dinitrophenol, etc.), human adjuvants such as Bacille Cahnette-GueYin and
CorynebacteriunZ
parvum, or similar immunostimulatory agents. If desired, the antibody
molecules directed
against ACETA can be isolated from the mammal (e.g., from the blood) and
further purified
by well known techniques, such as protein A chromatography to obtain the IgG
fraction.
The term "monoclonal antibody" or "monoclonal antibody composition", as used
herein, refers to a population of antibody molecules that contain only one
species of an antigen
binding site capable of immunoreacting with a particular epitope of ACETA. A
monoclonal
antibody composition thus typically displays a single binding affinity for a
particular ACETA
protein with which it immunoreacts. For preparation of monoclonal antibodies
directed
towards a particular ACETA protein, or derivatives, fragments, analogs or
homologs thereof,
any technique that provides for the production of antibody molecules by
continuous cell line
culture may be utilized. Such techniques include, but are not limited to, the
hybridoma
technique (see Kohler & Milstein, 1975 Natztj°e 256: 495-497); the
trioma technique; the
human B-cell hybridoma technique (see Kozbor, et al., 1983 Innnunol Today 4:
72) and the
EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et
al., 1985 In:
CA 02377768 2001-12-18
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MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
Human
monoclonal antibodies may be utilized in the practice of the present invention
and may be
produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci
USA 80:
2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro
(see Cole, et
al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc.,
pp. 77-96).
According to the invention, techniques can be adapted for the production of
single-chain antibodies specific to a ACETA protein (see e.g., U.S. Patent No.
4,946,778). In
addition, methods can be adapted for the construction of Fab expression
libraries (see e.g.,
Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective
identification of
monoclonal F~~ fragments with the desired specificity for a ACETA protein or
derivatives,
fragments, analogs or homologs thereof. Non-human antibodies can be
"humanized" by
techniques well known in the art. See e.g., U.S. Patent No. 5,225,539.
Antibody fragments
that contain the idiotypes to a ACETA protein may be produced by techniques
known in the
art including, but not limited to: (i) an F~ab.~, fragment produced by pepsin
digestion of an
antibody molecule; (ii) an Fav fragment generated by reducing the disulfide
bridges of an F~ab,o
fragment; (iii) an Fa,, fragment generated by the treatment of the antibody
molecule with papain
and a reducing agent and (iv) F~, fragments.
Additionally, recombinant anti-ACETA antibodies, such as chimeric and
humanized
monoclonal antibodies, comprising both human and non-human portions, which can
be made
using standard recombinant DNA techniques, are within the scope of the
invention. Such
chimeric and humanized monoclonal antibodies can be produced by recombinant
DNA
techniques known in the art, for example using methods described in PCT
International
Application No. PCT/US86/02269; European Patent Application No. 184,187;
European
Patent Application No. 171,496; European Patent Application No. 173,494; PCT
International
Publication No. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent
Application No.
125,023; Better et a1.(1988) Science 240:1041-1043; Liu et al. (1987) PNAS
84:3439-3443;
Liu et al. (1987) Jlmmunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218;
Nishimura
et al. (1987) Cancer Res 47:999-1005; Wood et al. (1985) Natatre 314:446-449;
Shaw et u1.
(1988) JNatl Cancerlnst. 80:1553-1559); Morrison(1985) Science 229:1202-1207;
Oi et al.
(1986) BioTeclzniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986)
Nature 321:552-525;
51
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Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) Jlmmunol
141:4053-4060.
In one embodiment, methods for the screening of antibodies that possess the
desired
specificity include, but are not limited to, enzyme-linked immunosorbent assay
(ELISA) and
other immunologically-mediated techniques known within the art. In a specific
embodiment,
selection of antibodies that are specific to a particular domain of a ACETA
protein is
facilitated by generation of hybridomas that bind to the fragment of a ACETA
protein
possessing such a domain. Antibodies that are specific for one or more domains
within a
ACETA protein, e.g., domains spanning the above-identified conserved regions
of ACETA
family proteins, or derivatives, fragments, analogs or homologs thereof, are
also provided
herein.
Anti-ACE TA antibodies may be used in methods known within the art relating to
the
localization and/or quantitation of a ACETA protein (e.g., for use in
measuring levels of the
ACETA protein within appropriate physiological samples, for use in diagnostic
methods, for
use in imaging the protein, and the like). In a given embodiment, antibodies
for ACETA
proteins, or derivatives, fragments, analogs or homologs thereof, that contain
the antibody
derived binding domain, are utilized as pharmacologically-active compounds
[hereinafter
"Therapeutics"].
An anti-ACETA antibody (e.g., monoclonal antibody) can be used to isolate
ACETA
by standard techniques, such as affinity chromatography or
immunoprecipitation. An
anti-ACETA antibody can facilitate the purification of natural ACETA from
cells and of
recombinantly produced ACETA expressed in host cells. Moreover, an anti-ACETA
antibody
can be used to detect ACETA protein (e.g., in a cellular lysate or cell
supernatant) in order to
evaluate the abundance and pattern of expression of the ACETA protein. Anti-
ACETA
antibodies can be used diagnostically to monitor protein levels in tissue as
part of a clinical
testing procedure, e.g., to, for example, determine the efficacy of a given
treatment regimen.
Detection can be facilitated by coupling (i.e., physically linking) the
antibody to a detectable
substance. Examples of detectable substances include various enzymes,
prosthetic groups,
fluorescent materials, luminescent materials, bioluminescent materials, and
radioactive
materials. Examples of suitable enzymes include horseradish peroxidase,
alkaline
phosphatase, -galactosidase, or acetylcholinesterase; examples of suitable
prosthetic group
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WO 01/02609 PCT/US00/40292
complexes include streptavidin/biotin and avidin/biotin; examples of suitable
fluorescent
materials include umbelliferone, fluorescein, fluorescein isothiocyanate,
rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an
example of a
luminescent material includes luminol; examples of bioluminescent materials
include
luciferase, luciferin, and aequorin, and examples of suitable radioactive
material include'''I,
1311, 3>s Or 3H.
ACETA RECOMBINANT EXPRESSION VECTORS AND HOST CELLS
Another aspect of the invention pertains to vectors, preferably expression
vectors,
containing a nucleic acid encoding ACETA protein, or derivatives, fragments,
analogs or
homologs thereof. As used herein, the term "vector" refers to a nucleic acid
molecule capable
of transporting another nucleic acid to which it has been linked. One type of
vector is a
"plasmid", which refers to a linear or circular double stranded DNA loop into
which additional
DNA segments can be ligated. Another type of vector is a viral vector, wherein
additional
DNA segments can be ligated into the viral genome. Certain vectors are capable
of
1 S autonomous replication in a host cell into which they are introduced
(e.g., bacterial vectors
having a bacterial origin of replication and episomal mammalian vectors).
Other vectors (e.g.,
non-episomal mammalian vectors) are integrated into the genome of a host cell
upon
introduction into the host cell, and thereby are replicated along with the
host genome.
Moreover, certain vectors are capable of directing the expression of genes to
which they are
operatively linked. Such vectors are referred to herein as "expression
vectors". In general,
expression vectors of utility in recombinant DNA techniques are often in the
form of plasmids.
In the present specification, "plasmid" and "vector" can be used
interchangeably as the plasmid
is the most commonly used form of vector. However, the invention is intended
to include such
other forms of expression vectors, such as viral vectors (e.g., replication
defective retroviruses,
adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of
the
invention in a form suitable for expression of the nucleic acid in a host
cell, which means that
the recombinant expression vectors include one or more regulatory sequences,
selected on the
basis of the host cells to be used for expression, that is operatively linked
to the nucleic acid
sequence to be expressed. Within a recombinant expression vector, "operably
linked" is
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CA 02377768 2001-12-18
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intended to mean that the nucleotide sequence of interest is linked to the
regulatory
sequences) in a manner that allows for expression of the nucleotide sequence
(e.g., in an in
vitro transcription/translation system or in a host cell when the vector is
introduced into the
host cell). The term "regulatory sequence" is intended to includes promoters,
enhancers and
other expression control elements (e.g., polyadenylation signals). Such
regulatory sequences
are described, for example, in Goeddel; GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Cali~ (1990). Regulatory sequences
include
those that direct constitutive expression of a nucleotide sequence in many
types of host cell
and those that direct expression of the nucleotide sequence only in certain
host cells (e.g.,
tissue-specific regulatory sequences). It will be appreciated by those skilled
in the art that the
design of the expression vector can depend on such factors as the choice of
the host cell to be
transformed, the level of expression of protein desired, etc. The expression
vectors of the
invention can be introduced into host cells to thereby produce proteins or
peptides, including
fusion proteins or peptides, encoded by nucleic acids as described herein
(e.g., ACETA
proteins, mutant forms of ACETA, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for
expression of
ACETA in prokaryotic or eukaryotic cells. For example, ACETA can be expressed
in
bacterial cells such as E. coli, insect cells (using baculovirus expression
vectors) yeast cells or
mammalian cells. Suitable host cells are discussed further in Goeddel, GENE
ExPRESSION
TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Cali~
(1990).
Alternatively, the recombinant expression vector can be transcribed and
translated in vitro, for
example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried OLIt in E. coli
with vectors
containing constitutive or inducible promoters directing the expression of
either fusion or
non-fusion proteins. Fusion vectors add a number of amino acids to a protein
encoded therein,
usually to the amino terminus of the recombinant protein. Such fusion vectors
typically serve
three purposes: (1) to increase expression of recombinant protein; (2) to
increase the solubility
of the recombinant protein; and (3) to aid in the purification of the
recombinant protein by
acting as a ligand in affinity purification. Often, in fusion expression
vectors, a proteolytic
cleavage site is introduced at the junction of the fusion moiety and the
recombinant protein to
enable separation of the recombinant protein from the fusion moiety subsequent
to purification
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CA 02377768 2001-12-18
WO 01/02609 PCT/US00/40292
of the fusion protein. Such enzymes, and their cognate recognition sequences,
include Factor
Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia
Biotech Inc; Smith and Johnson (1988) Gene 67:31-40), pMAL (New England
Biolabs,
Beverly, Mass.) and pRITS (Pharmacia, Piscataway, N.J.) that fuse glutathione
S-transferase
(GST), maltose E binding protein, or protein A, respectively, to the target
recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors include
pTrc
(Amrann et al., (1988) Gene 69:301-315) and pET l 1d (Studier et al., GENE
EXPRESSION
TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.
(1990)
60-89).
One strategy to maximize recombinant protein expression in E. coli is to
express the
protein in a host bacteria with an impaired capacity to proteolytically cleave
the recombinant
protein. See, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY
185,
Academic Press, San Diego, Cali~ (1990) 119-128. Another strategy is to alter
the nucleic
acid sequence of the nucleic acid to be inserted into an expression vector so
that the individual
codons for each amino acid are those preferentially utilized in E. coli (Wada
et al., (1992)
Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of
the invention
can be carried out by standard DNA synthesis techniques.
In another embodiment, the ACETA expression vector is a yeast expression
vector.
Examples of vectors for expression in yeast S. cerevisiae include pYepSecl
(Baldari, et al.,
(1987) EMBOJ6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943),
pJRY88
(Schultz et al., (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San
Diego, Calif.),
and picZ (InVitrogen Corp, San Diego, Cali~).
Alternatively, ACETA can be expressed in insect cells using baculovirus
expression
vectors. Baculovirus vectors available for expression of proteins in cultured
insect cells (e.g.,
SF9 cells) include the pAc series (Smith et al. (1983) Mol Cell Biol 3:2156-
2165) and the pVL
series (Lucklow and Summers (1989) Virology 170:31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in
mammalian
cells using a mammalian expression vector. Examples of mammalian expression
vectors
include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987)
EMBO J
CA 02377768 2001-12-18
WO 01/02609 PCT/US00/40292
6: 187-195). When used in mammalian cells, the expression vector's control
functions are
often provided by viral regulatory elements. For example, commonly used
promoters are
derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For
other
suitable expression systems for both prokaryotic and eukaryotic cells. See,
e.g., Chapters 16
and 17 of Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed.,
Cold
Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.,
1989.
In another embodiment, the recombinant mammalian expression vector is capable
of
directing expression of the nucleic acid preferentially in a particular cell
type (e.g.,
tissue-specific regulatory elements are used to express the nucleic acid).
Tissue-specific
regulatory elements are known in the art. Non-limiting examples of suitable
tissue-specific
promoters include the albumin promoter (liver-specific; Pinkert et al. (1987)
Genes Dev
1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv InzmurZOl
43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore
(1989) EMBO
J 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen
and
Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the
neurofilament
promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477), pancreas-specific
promoters
(Edlund et al. (1985) Science 230:912-916), and mammary gland-specific
promoters (e.g.,
milk whey promoter; U.S. Pat. No. 4,873,316 and European Application
Publication No.
264,166). Developmentally-regulated promoters are also encompassed, e.g., the
murine hox
promoters (Kessel and Gruss (1990) Science 249:374-379) and the oc-fetoprotein
promoter
(Campes and Tilghman (1989) Genes Dev 3:537-546).
The invention further provides a recombinant expression vector comprising a
DNA
molecule of the invention cloned into the expression vector in an antisense
orientation. That
is, the DNA molecule is operatively linked to a regulatory sequence in a
manner that allows
for expression (by transcription of the DNA molecule) of an RNA molecule that
is antisense to
ACETA mRNA. Regulatory sequences operatively linked to a nucleic acid cloned
in the
antisense orientation can be chosen that direct the continuous expression of
the antisense RNA
molecule in a variety of cell types, for instance viral promoters and/or
enhancers, or regulatory
sequences can be chosen that direct constitutive, tissue specific or cell type
specific expression
of antisense RNA. The antisense expression vector can be in the form of a
recombinant
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plasmid, phagemid or attenuated virus in which antisense nucleic acids are
produced under the
control of a high efficiency regulatory region, the activity of which can be
determined by the
cell type into which the vector is introduced. For a discussion of the
regulation of gene
expression using antisense genes see Weintraub et al., "Antisense RNA as a
molecular tool for
genetic analysis," Reviews--Trends in Genetics, Vol. 1(1) 1986.
Another aspect of the invention pertains to host cells into which a
recombinant
expression vector of the invention has been introduced. The terms "host cell"
and
"recombinant host cell" are used interchangeably herein. It is understood that
such terms refer
not only to the particular subject cell but also to the progeny or potential
progeny of such a
cell. Because certain modifications may occur in succeeding generations due to
either
mutation or environmental influences, such progeny may not, in fact, be
identical to the parent
cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, ACETA
protein
can be expressed in bacterial cells such as E. coli, insect cells, yeast or
mammalian cells (such
as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells
are known to
those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via
conventional
transformation or transfection techniques. As used herein, the terms
"transformation" and
"transfection" are intended to refer to a variety of art-recognized techniques
for introducing
foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate
or calcium
chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or
electroporation.
Suitable methods for transforming or transfecting host cells can be found in
Sambrook, et al.
(MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor
Laboratory,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and
other laboratory
manuals.
For stable transfection of mammalian cells, it is known that, depending upon
the
expression vector and transfection technique used, only a small fraction of
cells may integrate
the foreign DNA into their genome. In order to identify and select these
integrants, a gene that
encodes a selectable marker (e.g., resistance to antibiotics) is generally
introduced into the host
cells along with the gene of interest. Various selectable markers include
those that confer
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resistance to drugs, such as 6418, hygromycin and methotrexate. Nucleic acid
encoding a
selectable marker can be introduced into a host cell on the same vector as
that encoding
ACETA or can be introduced on a separate vector. Cells stably transfected with
the introduced
nucleic acid can be identified by drug selection (e.g., cells that have
incorporated the selectable
marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in
culture, can
be used to produce (i.e., express) an ACETA protein. Accordingly, the
invention further
provides methods for producing ACETA protein using the host cells of the
invention. In one
embodiment, the method comprises culturing the host cell of invention (into
which a
recombinant expression vector encoding ACETA has been introduced) in a
suitable medium
such that ACETA protein is produced. In another embodiment, the method further
comprises
isolating ACETA from the medium or the host cell.
PHARMACEUTICAL COMPOSITIONS
The ACETA nucleic acid molecules, ACETA proteins, and anti-ACETA antibodies
(also referred to herein as "active compounds") of the invention, and
derivatives, fragments,
analogs and homologs thereof, can be incorporated into pharmaceutical
compositions suitable
for administration. Such compositions typically comprise the nucleic acid
molecule, protein, or
antibody and a pharmaceutically acceptable carrier. As used herein,
"pharmaceutically
acceptable carrier" is intended to include any and all solvents, dispersion
media, coatings,
antibacterial and antifungal agents, isotonic and absorption delaying agents,
and the like,
compatible with pharmaceutical administration. Suitable carriers are described
in the most
recent edition of Remington's Pharmaceutical Sciences, a standard reference
text in the field,
which is incorporated herein by reference. Preferred examples of such carriers
or diluents
include, but are not limited to, water, saline, finger's solutions, dextrose
solution, and 5%
human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may
also be
used. The use of such media and agents for pharmaceutically active substances
is well known
in the art. Except insofar as any conventional media or agent is incompatible
with the active
compound, use thereof in the compositions is contemplated. Supplementary
active compounds
can also be incorporated into the compositions.
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A pharmaceutical composition of the invention is formulated to be compatible
with its
intended route of administration. Examples of routes of administration include
parenteral, e.g.,
intravenous, intradermal; subcutaneous, oral (e.g., inhalation), transdermal
(topical),
transmucosal, and rectal administration. Solutions or suspensions used for
parenteral,
intradermal, or subcutaneous application can include the following components:
a sterile
diluent such as water for injection, saline solution, fixed oils, polyethylene
glycols, glycerine,
propylene glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate;
chelating agents such
as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or
phosphates, and agents
for the adjustment of tonicity such as sodium chloride or dextrose. The pH can
be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation
can be enclosed in ampoules, disposable syringes or multiple dose vials made
of glass or
plastic.
Pharmaceutical compositions suitable for injectable use include sterile
aqueous
solutions (where water soluble) or dispersions and sterile powders for the
extemporaneous
preparation of sterile injectable solutions or dispersion. For intravenous
administration,
suitable carriers include physiological saline, bacteriostatic water,
Cremophor ELTM (BASF,
Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the
composition must be
sterile and should be fluid to the extent that easy syringeability exists. It
must be stable under
the conditions of manufacture and storage and must be preserved against the
contaminating
action of microorganisms such as bacteria and fungi. The carrier can be a
solvent or dispersion
medium containing, for example, water, ethanol, polyol (for example, glycerol,
propylene
glycol, and liquid polyethylene glycol, and the like), and suitable mixtures
thereof. The proper
fluidity can be maintained, for example, by the use of a coating such as
lecithin, by the
maintenance of the required particle size in the case of dispersion and by the
use of surfactants.
Prevention of the action of microorganisms can be achieved by various
antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic
acid, thimerosal, and
the like. In many cases, it will be preferable to include isotonic agents, for
example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
Prolonged
absorption of the injectable compositions can be brought about by including in
the
composition an agent which delays absorption, for example, aluminum
monostearate and
gelatin.
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Sterile injectable solutions can be prepared by incorporating the active
compound (e.g.,
a ACETA protein or anti-ACETA antibody) in the required amount in an
appropriate solvent
with one or a combination of ingredients enumerated above, as required,
followed by filtered
sterilization. Generally, dispersions are prepared by incorporating the active
compound into a
sterile vehicle that contains a basic dispersion medium and the required other
ingredients from
those enumerated above. In the case of sterile powders for the preparation of
sterile injectable
solutions, methods of preparation are vacuum drying and freeze-drying that
yields a powder of
the active ingredient plus any additional desired ingredient from a previously
sterile-filtered
solution thereof.
Oral compositions generally include an inert diluent or an edible earner. They
can be
enclosed in gelatin capsules or compressed into tablets. For the purpose of
oral therapeutic
administration, the active compound can be incorporated with excipients and
used in the form
of tablets, troches, or capsules. Oral compositions can also be prepared using
a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is applied
orally and swished
and expectorated or swallowed. Pharmaceutically compatible binding agents,
and/or adjuvant
materials can be included as part of the composition. The tablets, pills,
capsules, troches and
the like can contain any of the following ingredients, or compounds of a
similar nature: a
binder such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as
starch or lactose, a disintegrating agent such as alginic acid, Primogel, or
corn starch; a
lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal
silicon dioxide; a
sweetening agent such as sucrose or saccharin; or a flavoring agent such as
peppermint, methyl
salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of
an aerosol
spray from pressured container or dispenser which contains a suitable
propellant, e.g., a gas
such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For
transmucosal or transdermal administration, penetrants appropriate to the
barrier to be
permeated are used in the formulation. Such penetrants are generally known in
the art, and
include, for example, for transmucosal administration, detergents, bile salts,
and fusidic acid
derivatives. Transmucosal administration can be accomplished through the use
of nasal sprays
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or suppositories. For transdermal administration, the active compounds are
formulated into
ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with
conventional
suppository bases such as cocoa butter and other glycerides) or retention
enemas for rectal
delivery.
In one embodiment, the active compounds are prepared with carriers that will
protect
the compound against rapid elimination from the body, such as a controlled
release
formulation, including implants and microencapsulated delivery systems.
Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides,
polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for
preparation of
such formulations will be apparent to those skilled in the art. The materials
can also be
obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
Liposomal
suspensions (including liposomes targeted to infected cells with monoclonal
antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers. These can
be prepared
according to methods known to those skilled in the art, for example, as
described in U.S. Pat.
No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in
dosage
unit form for ease of administration and uniformity of dosage. Dosage unit
form as used herein
refers to physically discrete units suited as unitary dosages for the subject
to be treated; each
unit containing a predetermined quantity of active compound calculated to
produce the desired
therapeutic effect in association with the required pharmaceutical earner. The
specification for
the dosage unit forms of the invention are dictated by and directly dependent
on the unique
characteristics of the active compound and the particular therapeutic effect
to be achieved, and
the limitations inherent in the art of compounding such an active compound for
the treatment
of individuals.
The nucleic acid molecules of the invention can be inserted into vectors and
used as
gene therapy vectors. Gene therapy vectors can be delivered to a subject by,
for example,
intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or
by stereotactic
injection (see e.g., Chen et al. (1994) PNAS 91:3054-3057). The pharmaceutical
preparation of
the gene therapy vector can include the gene therapy vector in an acceptable
diluent, or can
comprise a slow release matrix in which the gene delivery vehicle is imbedded.
Alternatively,
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where the complete gene delivery vector can be produced intact from
recombinant cells, e.g.,
retroviral vectors, the pharmaceutical preparation can include one or more
cells that produce
the gene delivery system.
The pharmaceutical compositions can be included in a container, pack, or
dispenser
together with instructions for administration.
KITS AND NUCLEIC ACID COLLECTIONS FOR IDENTIFYING ACETA NUCLEIC ACIDS
In another aspect, the invention provides a kit useful for examining
hepatotoxicity of
agents. The kit can include nucleic acids that detect two or more ACETA
sequences. In
preferred embodiments, the kit includes reagents which detect 3, 4, 5, 6, 8,
10, 12, 15, 20, 25,
50, 100 or all of the ACETA nucleic acid sequences.
The invention also includes an isolated plurality of sequences which can
identify one or
more ACETA responsive nucleic acid sequences.
The kit or plurality may include, e.g., sequence homologous to ACETA nucleic
acid
sequences, or sequences which can specifically identify one or more ACETA
nucleic acid
sequences.
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in
conjunction with
the detailed description thereof, the foregoing description is intended to
illustrate and not limit
the scope of the invention, which is defined by the scope of the appended
claims. Other
aspects, advantages, and modifications are within the scope of the following
claims.
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