# Patent

## Title
Method of identifying toxic agents using NSAID-induced differential gene expression in liver

## Abstract

The invention provides methods of identifying toxic agents, e.g., hepatotoxic agents, using differential gene expression. Also provided are methods of predicting the risk level and or injury type of NSAIDs. Also disclosed are novel nucleic acid sequences whose expression is differentially regulated by NSAIDs.

## Background
RELATED APPLICATIONS
This application claims priority to U.S. Pat. No. 60/166,923, filed Nov. 22, 1999, and U.S. Pat. No. 60/183,531, filed Feb. 18, 2000, the disclosures of which are hereby incorporated by reference herein.


FIELD OF THE INVENTION
The invention relates generally to nucleic acids and polypeptides, and more particularly to the identification of differentially expressed nucleic acids and proteins in liver.
BACKGROUND OF THE INVENTION
Liver is the primary organ for biotransformation of chemical compounds and their detoxification. Liver injury produced by chemicals has been recognized for over 100 years, and hepatic damage is one of the most common toxicities among drugs at pre-clinical and clinical stages of drug development. Over 30% of new chemical entities (NCE) are generally terminated due to adverse liver effects in humans. During a period of 30 years, hepatotoxicity has been the major cause of drug withdrawal for safety reasons at the marketing stage, accounting for 18% overall drug withdrawal. Many of the drugs that are withdrawn from market due to hepatotoxicity produce lethality in a small percentage of patient population and are classified as type II lesion (or idiosyncratic, sporadic) toxicity.
Non-steroidal anti-inflammatory drugs (NSAIDs) are a group of unrelated chemical compounds that have been used to successfully treat rheumatic and musculospastic disease. Unfortunately, unwanted hepatotoxic side effects have lead to the premature market withdrawal of several NSAIDs, including Cincophen, Benoxaprofen, Piroxicam, Suprofen, and Bromfenac. The pervasiveness of idiosyncratic reactions of many NSAIDs has lead the Food and Drug Administrations Arthritis Advisory Board to conclude that NSAIDs as a group should be considered to induce hepatotoxicity.
It is estimated that annual NSAIDS consumption in the U.S exceeds 10,000 tons. Due to this large consumption of NSAIDS for a wide variety of pain and inflammatory conditions, it has become an important class of drugs responsible for liver injury, despite the overall extremely low incidence of producing hepato-toxicity. Liver injury resulting from NSAIDs can have several forms, including acute toxicity resulting from hepatocellular (parenchymal) damage (e.g. necrosis) and arrested bile flow (cholestasis). The general mechanism that is thought to mediate NSAIDS toxicity is idisyncratic reaction (Type II) to the drug (both immunologic and metabolic), which is dose independent, and presumably results from interindividual variation in drug metabolism. Currently no clear mechanism of drug-induced idiosyncratic toxicity is available. Accordingly, there remains a great need to elucidate the molecular basis of idiosyncratic hepatoxicity, such as NSAID-induced toxicity, including the identification of genes and proteins differentially expressed in response to administration of such drugs.
SUMMARY OF THE INVENTION
In accordance with the present invention, there are provided methods of screening and identifying test agents which induce hepatotoxicity, e.g. idiosyncratic hepatotoxicity. The methods of the invention are based in part on the discovery that certain nucleic acids are differentially expressed in liver tissue of animals treated with NSAIDs. These differentially expressed nucleic acids include novel sequences that, while previously described, have not heretofore been identified as responsive to drugs, such as NSAIDs, which induce idiosyncratic hepatoxicity.
In various aspects, the invention includes a method of screening a test agent for toxicity, e.g., idiosyncratic 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 drugs, e.g. NSAIDs, which induce idiosyncratic hepatotoxicity, 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 one aspect, expression in the test cell population is compared to the expression of a reference cell population exposed to a NSAID that is classified as low risk, very low risk, or overdose risk of hepatoxicity, thereby to predict whether the test agent has low, very low, or overdose risk of hepatoxicity. In another aspect, the test cell population is compared to the expression of a reference cell population exposed to a NSAID which induces a known type of hepatic injury, e.g. hepatocellular damage, cholestasis, or elevated transaminase level, thereby to predict whether the test agent is likely to induce a given type of hepatoxic injury.
In a further aspect, the invention provides a method of assessing the hepatotoxicity, e.g. idiosyncratic 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 NSAID-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.
Also provided are novel nucleic acids, as well as their encoded polypeptides, whose expression is responsive to the effects of NSAID.
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.


DETAILED DESCRIPTION OF THE INVENTION
The invention is based in part on the discovery of nucleic acid sequences which are differentially expressed in rodent liver cells upon administration of NSAIDS. The discovery includes groups of nucleic acid sequences whose expression is correlated with hepatotoxicity risk associated with, and injury type induced by, NSAID administration.
The differentially expressed nucleic acid sequences were identified by examining 29 different NSAIDS that have varying degrees of hepatotoxicity. These 29 drugs, shown in Table 1, below, were first categorized as low dose (1-75 mg/kg) and high dose (above 75 mg/kg) drugs, then classified as non-toxic, toxic, and those withdrawn from market (within each dose). Each of the 29 NSAIDS was administered orally to groups (3 animals per group) of 12 week old male Sprague Dawley rats for 72 hours (3 days) at the dosages specified in Table 1 (e.g. Naproxen: 54 mg/kg/day PO in QD×3 days in H2O). Vehicle controls (water, ethanol, canola oil) were also included (3 animals per group). The animals were sacrificed 24 hours after the final dose, liver tissue was removed on necroscopy, and total RNA was recovered from the dissected tissue. Complementary DNA (cDNA) was prepared and samples were processed through GENECALLINGTM differential expression analysis, as described in U.S. Pat. No. 5,871,697 and in Shimkets et al., Nature Biotechnology 17: 798-803 (1999), the disclosures of which are hereby incorporated by reference herein.







TABLE 1







NSAIDS and Dosages Administered









Compound
Dose
Vehicle













Acetaminophen
133
mg/kg/day p.o. in QD × 3 days.
10% Ethanol





Vehicle


Acetylsalicylic
200
mg/kg/day p.o. in QD × 3 days.
Canola Oil


Acid


Vehicle


Benoxaprofen
16
mg/kg/day p.o. in QD × 3 days.
H2O Vehicle


Bromfenac
7.5
mg/kg/day p.o. in QD × 3 days.
H2O Vehicle


Celecoxib
89
mg/kg/day p.o. in QD × 3 days.
Canola Oil





Vehicle


Diclofenac
38
mg/kg/day p.o. in QD × 3 days.
H2O Vehicle


Etodolac
30
mg/kg/day p.o. in QD × 3 days.
10% Ethanol





Vehicle


Felbinac
33
mg/kg/day p.o. in QD × 3 days.
Canola Oil





Vehicle


Fenoprofen
154
mg/kg/day p.o. in QD × 3 days.
Canola Oil





Vehicle


Flurbiprofen
10
mg/kg/day p.o. in QD × 3 days.
Canola Oil





Vehicle


Ibuprofen
211
mg/kg/day p.o. in QD × 3 days.
H2O Vehicle


Indomethacin
4
mg/kg/day p.o. in QD × 3 days.
Canola Oil





Vehicle


Ketoprofen
10
mg/kg/day p.o. in QD × 3 days.
Canola Oil





Vehicle


Ketorolac
1.5
mg/kg/day p.o. in QD × 3 days.
10% Ethanol





Vehicle


Meclofenamate
20
mg/kg/day p.o. in QD × 3 days.
H2O Vehicle


Mefenamic Acid
79
mg/kg/day p.o. in QD × 3 days.
Canola Oil





Vehicle


Nabumetone
143
mg/kg/day p.o. in QD × 3 days.
Canola Oil





Vehicle


Naproxen
54
mg/kg/day p.o. in QD × 3 days.
10% Ethanol





Vehicle


Olsalazine
222
mg/kg/day p.o. in QD × 3 days.
H2O Vehicle


Oxaprozin
100
mg/kg/day p.o. in QD × 3 days.
Canola Oil





Vehicle


Phenacetin
100
mg/kg/day p.o. in QD × 3 days
Canola Oil





Vehicle


Phenylbutazone
100
mg/kg/day p o. in QD × 3 days.
Canola Oil





Vehicle


Piroxicam
20
mg/kg/day p.o. in QD × 3 days.
Canola Oil





Vehicle


Sulindac
77
mg/kg/day p.o. in QD × 3 days.
Canola Oil





Vehicle


Sulphusalazine
338
mg/kg/day p.o. in QD × 3 days.
Canola Oil





Vehicle


Suprofen
20
mg/kg/day p.o. in QD × 3 days.
Canola Oil





Vehicle


Tenoxicam
10
mg/kg/day p.o. in QD × 3 days.
Canola Oil





Vehicle


Tolmentin
100
mg/kg/day p.o. in QD × 3 days.
H2O Vehicle


Zomepirac
19
mg/kg/day p.o. in QD × 3 days.
Canola Oil





Vehicle









3635 gene fragments were initially found to be differentially expressed in rat liver tissue (analysis of variance, p<0.01) in response to these compounds. The compounds were then classifed according to hepatotoxicity risk, as indicated in Table 2.







TABLE 2







Hepatotoxicity Risk of NSAIDs










Compound
Risk







Acetaminophen
Overdose Risk



Acetylsalicylic Acid
Overdose Risk



Benoxaprofen
Low Risk



Bromfenac
Low Risk



Celecoxib
Unknown



Diclofenac
Low Risk



Etodolac
Very Low Risk



Felbinac
Unknown



Fenoprofen
Very Low Risk



Flurbiprofen
Very Low Risk



Ibuprofen
Very Low Risk



Indomethacin
Very Low Risk



Ketoprofen
Very Low Risk



Ketorolac
Unknown



Meclofenamate
Very Low Risk



Mefenamic Acid
Very Low Risk



Nabumetone
Very Low Risk



Naproxen
Very Low Risk



Olsalazine
Unknown



Oxaprozin
Very Low Risk



Phenacetin
Overdose Risk



Phenylbutazone
Low Risk



Piroxicam
Very Low Risk



Sulindac
Low Risk



Sulphasalazine
Unknown



Suprofen
Very Low Risk



Tenoxicam
Very Low Risk



Tolmentin
Very Low Risk



Zomepirac
Very Low Risk










In order to discriminate among these groups, the above compound set was divided into a training set (consisting of three compounds per group), and a test set (consisting of the remainder. This was done to minimize the reliance on the assumptions required for parametric analyses. Compounds with unknown risk were not used in this analysis. The training set employed is shown in Table 3.







TABLE 3







Training Set of NSAIDs by Risk Classification










Control
Low Risk
Very Low Risk
Overdose Risk





Sterile water
Benoxaprofen
Flurbiprofen
Acetaminophen


10% Ethanol
Phenylbutazone
Oxaprozin
Acetylsalicylic Acid


Canola oil
Sulindac
Tenoxicam
Phenacetin









The 3635 differentially expressed nucleic acid fragments were then analyzed using a stepwise multivariate analysis of variance as follows:
1. Calculate 3635 T2 (yi1) (Hoettelling's trace, one of the test statistics used for this analysis) values, one for each differentially expressed fragment. The fragment with the largest individual T2 value is selected as the first discriminatory set (yi1).
2. Calculate 3634 T2 (yi 1,yi2) values, one for each combination of two fragments. The fragment pair with the largest individual T2 value are selected as the second discriminatory set.
3. Calculate 3626 T2 (yi1,yi2,yi3, . . . yi,10), one for each combination often fragments.
The fragment set with the largest T2 value is selected as the final discriminatory set.
This stepwise procedure is used whenever the number of dependent variables (gene fragments) exceeds the number of independent variables (samples). In addition to fragment addition, fragment elimination occurs whenever an added fragment no longer contributes significant discriminatory power to the existing set. This eliminates bias as to the order fragments enter the model (Ahrens and Läuter, Mehrdimensionale Varianzanalyse, Akademie-Verlag, Berlin (1974); Dziuda, Medical Inform. 15(4): 319 (1990)).
This analysis protocol identified ten fragments that significantly (p=6.02×10−28) discriminated among the drugs in the test set. Two fragments on this list were not required to maintain the discriminatory ability and were subsequently removed (p=3.96×10−26). Differential expression of these gene fragments were successfully confirmed using an unlabeled oligonucleotide competition assay (Shimkets et al., Nature Biotechnology 17: 198-803 (1999)). The 8 fragments (RISKMARKER 1-8) represent both novel and known rat genes for which the sequence identity to genes in public databases is either high (>90%), moderate (70-90%), or low (<70%).
The identity of these 8 hepatoxicity risk discriminatory nucleic acid sequences(with GenBank accession numbers) are further described below. Where appropriate, the cloned sequence from isolation is provided; this sequence was then extended using either Genbank rat ESTs or from internally (Curagen Corporation) sequenced rat fragments. The extended contig sequence is provided as “consensus.” Finally, the best BlastN and BlastX results are also provided. In some instances the cloned sequence is identical to a known rat gene, in those instances the name of the gene and a database accession number and the sequence listed in the database is provided:
RISKMARKER 1
RISKMARKER 1 is a novel 1265 bp gene fragment, which has 67% sequence identity to human rac1 genomic fragment [AJ132695], probable 3′ UTR. The nucleic acid was initially identified in a cloned fragment having the following sequence:










  1
caattgaaaa aagtttgttc tagtggtcga aaggcccaac actgtgttct tgccagtgag
 (SEQ ID NO: 1)



 61
ttaggttgta cagaacggcg ttagcactag cgcttgacag aacctcacag acccaaaggt


121
acc






The cloned sequence was assembled into a contig resulting in the following consensus sequence:










   1
TTTTTTTTTTTTTTTTTCAAGTTCCAAAGACATTTTTTTTTTTTTTTTTATGATTCAAGGATTTATTAAGTCATACATGC
  (SEQ ID NO: 2)



  81
AAAACATACTGCTAACTGCATTAGCAAAAGATCAATGTAAAAACACTCCACAATTCTGCAACTGTCAATTGAAAAAAGTT


 161
TGTTCTAGTGGTCGAAAGGCCCAACACTGTGTTCTTGCCAGTGAGTTAGGTTGTACAGAACGGCGTTAGCACTAGCGCTT


 241
GACAGAACCTCACAGACCCAAAGGTACCGGAAGCATGTGTCCGCGTGGGTGAGGTCTAGAGGGGGCGGCATCAATCACAT


 321
GACAGTGTTGGTACTCTGGCAAGACAGTGATGTTTCAGAATATCTAAAATAGTTTAAAAACTGTAAAGCCGCAGCACGTG


 401
ATTTCTACACCCAGTTACTAGAAAACGAAGGGAAGCACTAGTCAGCTGAGTAAAGGAAGGTGAAAACAGGAACGCACTTC


 481
TACTATCTACCAAAAAAATCTCCGAATGCATTATCAGAAAGATCTTATAGTACAGGTCAGACATATTGCTCGTTAAGAAG


 561
GGGGTCCTAAAGAAAAGCACTTGCTAAGTTAGCAACTGTGAGGATGGCCAGTTTAAATATGGACTCAACGCCCCATCTGG


 641
GGAGGGACAGCAGGGGGAAGGGGGGCTCAAGAGAGACACTGATAAGATCGGCCATTTGTCATCTACTGTTTGACAGAAAT


 721
TAACCGTTAAAAAGCTTTACCCGTGACACTTTTATTCAGTTGAATTACTCCATGTACAATGTAGTGTAAATTAATCTCTA


 801
CTTCATATTAGTCAAAATACTGTCTGTCTCCTTTGATGACGTCGTGTTTCACACACTCCACCCAGCACACCCACGACTAG


 881
GAACAGAATACTTCGTTAGAGGCAACACAGGAGCCAGAGTTCTGTTCAAAGCCTGCAGAAGCCGGTCAGCTGGTATTTTA


 961
GAGAACTCACTATGAAATCAAAGAGCAGAGCTGTTACACCCATCGTGACGTACAGTACAAAGTTACGTAATGAGCATGGG


1041
CTGATAAGTTACAGGTGCGTTACATGGCAGCGTGTCATTAAGGAGGCTGTGCTGTGTCACACGGTCTGGGAGCTACGGGA


1121
GGGTCTGCACCCCTGAGCCCAGAAGCTGCAGTCTTCTTAAGGACAAAGTCTCTCAACAGCTTAGTGCTTACGTGTTCTCA


1201
GCACAACGCAACTTAGTTCACAAGGTATTTTGGCAATTCTTAATCTGAGCAAGAATAGGGGATTTT






Blast-N Results:

>gb: GENBANK-ID: HSA132695|acc:AJ132695 Homo sapiens rac1 gene—Homo sapiens, 28567 bp.

Top Previous Match Next Match

    
    
        Length=28,567
        Minus Strand HSPs:
    
    










Score = 1328 (199.3 bits), Expect = 2.8e-90, Sum P(2) = 2.8e-90



Identities = 694/1022 (67%), Positives = 694/1022 (67%), Strand = Minus/Plus












Query:
 1261
CCCCTATTCTTGCTCAGATTAAGAATTGCCAAAATACCTTGTGAACTAAGTTGC---GTT
1205






|||| ||||||| ||||||||||| ||||||||||||||| |||||||   |||   |||


Sbjct:
26871
CCCCCATTCTTGTTCAGATTAAGAGTTGCCAAAATACCTTCTGAACTACACTGCATTGTT
26930


Query:
 1204
GTGCTGAGAACACGTAAGCACTAAGCTGTTG-AGAGACTTT-GTCCTTAAGAAGACTGCA
1147




|||| ||||||||  | ||||| | || ||| | |||| || ||| || ||||||| | |


Sbjct:
26931
GTGCCGAGAACACCGA-GCACTGAACT-TTGCAAAGACCTTCGTCTTTGAGAAGACGGTA
26988


Query:
 1146
GCTTCTGGGCTCAGG-GGTGCAGACCCTCCCGTAGCTCCCAGACCGTGTGACACAGCACA
1088




|||||||   | ||| ||||||||| ||  | |  |||| |    || |  | ||| |


Sbjct:
26989
GCTTCTGCAGTTAGGAGGTGCAGACACTTGC-T--CTCCTATGTAGT-T--CTCAG-AT-
27040


Query:
 1087
GCCTCCTTAATGACACGCTGCC--ATGTAACGCA-CCTGT-AACTTATCAGCCCAT--GC
1034




|| |     |  |||  || ||  ||| | ||   ||  | |||| | |||    |  ||


Sbjct:
27041
GCGTAAAGCAGAACAGCCTCCCGAATGAAGCGTTGCCATTGAACTCACCAGTGAGTTAGC
27100


Query:
 1033
T-CA--T-TACGTAACTTTGTACTGTAC-GTCACGATGG-GTGTAACAGCTCTGCTCTTT
980




  ||  | | |   || |   | ||||| || |  |||| ||| | |    | ||||


Sbjct:
27101
AGCACGTGTTCCCGACATAACATTGTACTGT-A--ATGGAGTG-AGCGTAGCAGCTCAGC
27156


Query:
  979
GATTTCATAGTGAGT-TCTCTAAAATACCAGCTGACCGGCTTCTG-CAGGCTTT-GAACA
923




  |||   | | ||| | | | |  |   ||| ||  |  ||||| |  G|||| |   |


Sbjct:
27157
TCTTTGG-A-TCAGTCTTTGTGATTTCATAGC-GA--GTTTTCTGACCAGCTTTTGCGGA
27211


Query:
  922
GAACTCTGG-CTC--CTG-TGTTGCCTCTAACGAAGTATTCTGTTCCTAGTCGTGGGTGT
867




||  | ||  |    ||| | || ||||||| |||| ||||||||  |||  ||||||||


Sbjct:
27212
GATTT-TGAACAGAACTGCTATTTCCTCTAATGAAGAATTCTGTT--TAGCTGTGGGTGT
2768


Query:
  866
GCTGGGTGGAGTGTGTGAAACACGACGTCATCAAAGGAGACAGACAGTATTTTGACTAA-
808




|| |||||| |||||||      ||     |||||||| | ||||||||||||||| ||


Sbjct:
27269
GCCGGGTGGGGTGTGTGT-----GA-----TCAAAGGACAAAGACAGTATTTTGACAAAA
27318


Query:
  807
TATGAAGTAGAGATTAATT-TACACT--ACATTGTACATGGAGTAAT-TCAACTGAATAA
752




|| ||||| |||||| |   |||| |  |||  | | ||| |  | | |||   | | ||


Sbjct:
27319
TACGAAGTGGAGATTTACACTACATTGTACAAGG-A-ATGAA--AGTGTCACGGGTA-AA
27373


Query:
  751
AAGTGTCACGGGTAAAGCTTTTT-AACGGTTAATTTCTG-TCAAACAGTAGATGACAA-A
695




|| | | |  ||| ||  | | | ||  |  | |   || | ||| |   | ||  |  |


Sbjct:
27374
AACTCTAAAAGGTTAATTTCTGTCAAATGC-AGTAGATGATGAAAGAAAGGTTGGTATTA
27432


Query:
  694
TG-GCCGATCTTATCAGTGTCTCTCTTGAGCCCCCCTTCCCCCTGCTGTCCCTCCCCAGA
636




|  |   || || ||     || | ||     | | || | |||||  | |||||||| |


Sbjct:
27433
TCAGGAAATGTTTTCTTAAGCT-T-TTCCTTTCTC-TTACACCTGCCATGCCTCCCCAAA
27489


Query:
  635
TGGGGCGTTGAGTCCATATTTAAACTGGCCATCCTCACAGTTGCTAACTTAGCAAGTGCT
576




| |||| || | | ||| ||||||||||   | ||   ||| |||||||||| |||||||


Sbjct:
27490
TTGGGCATTTAATTCATCTTTAAACTGGTTGTTCTGTTAGTCGCTAACTTAGTAAGTGCT
27549


Query:
  575
TTTCTT-TAGGACCCCCTTCTTAACGAGCAATATGTCTGACCTGTACTATAAGATCTTTC
517




|||||| ||| ||||| |||| |  |||||||||| ||  | |||| ||||| |||||||


Sbjct:
27550
TTTCTTATAGAACCCC-TTCTGACTGAGCAATATGCCTC-CTTGTATTATAAAATCTTTC
27607


Query:
  516
TGATAATGCATTCGGAGATTTTTTTGGTAGATAGTAGAAGTGCGTTCC-TGTTTTCACCT
458




|||||||||||| | || ||||||||     ||||| |||||| |||| ||||      |


Sbjct:
27608
TGATAATGCATTAGAAGGTTTTTTTGTCGATTAGTAAAAGTGCTTTCCATGTTACTTTAT
27667


Query:
  457
TCCTT--TACTCAG--CTGAC-T-AGTGCTTCCCTTCGTTT--TCTAGTAA-C-TGGGT-
409




T|     T| T |G  |T  | T |GT  TT|   T   T   T T|  || | TG GT


Sbjct:
27668
TCAGAGCTAATAAGTGCTTTCCTTAGTT-TTCTAGTAACTAGGTGTAAAAATCATGTGTT
27726


Query:
  408
GTAGAAATCACGTGCTGCGGCT-TTACAG-T--TT-TTAAACTATTTTAGATATTCTGAA
354




G |G   T   GT  T     T TT  |G T  TT TT||||T|T   | |  TT|T ||


Sbjct:
27727
GCAGCTTTATAGTTTTTAAAATATTTTAGATAATTCTTAAACTATG--A-ACCTTCTTAA
27783


Query:
  353
ACATCACTGTCTTGCCAGAGTACCAACACTGTCATGTGATTGATGCCGCCCCCTCTAGAC
294




 ||T|||TGT|TTG|||G| T||| ||||TGT||  TG|   |T | G ||| T|T  ||


Sbjct:
27764
-CATCACTGTCTTGCCAGATTACCGACACTGTCACTTGACCAATACTGACCC-TCTTTAC
27841


Query:
  293
CTCACCCACGCGGACACATGCTTCCGGTA--C-CTTTGGGTCTGTGAGGTTC
245
(SEQ ID NO: 15)




|T| |||||G|GG||||| G| T|| GT|  | |TTTG  T T TG| GTT|


Sbjct:
27842
CTCGCCCACGCGGACACACGCCTCCTGTAGTCGCTTTGCCTAT-TGATGTTC
27892
(SEQ ID NO: 16)








Score = 930 (139.5 bits), Expect = 2.8e-90, Sum P(2) = 2.8e-90



Identities = 270/354 (76%), Positives = 270/354 (76%), Strand = Minus/Plus












Query:
  357
TGAAACATCACTGTCT-TGCCAGAGTACCAACACTGTCATGTGATTGATGCCGCCCCCTC
299






||  | || || | |  || ||    ||||| |||| |   |  || |   ||||| | |


Sbjct:
27795
TGCCAGATTACCGACACTGTCACTTGACCAATACTGACCC-TCTTT-ACCTCGCCCACGC
27852


Query:
  298
TAGAC-CTCACCCAC-GCGGACACAT-GCTTCC-G--GTACCTTTGGGTCTGTGAGGTTC
245




  ||| | | ||  | |  | | | | || |   |  || ||||||||||||||||||||


Sbjct:
27853
G-GACACCGCCTCCT GT|GTCGCTTTGCCTATTGATGTTCCTTTGGGTCTGTGAGGTTC
27911


Query:
  244
TGTCAA--GCGCTAGTGCTAACGCCGTTCTGTACAACCTAACTCACTGGCAAGAACACAG
187




TGT AA  G G|TAGTG|T A|G  GTT|TGTA|AA| TAA|T|A|TGG| AGAA A|AG


Sbjct:
27912
TGTAAACTGTGCTAGTGCTGACGATGTTCTGTACAACTTAACTCACTGGCGAGAATACAG
27971


Query:
  186
TGTTGGGCCTTTCGACCACTAGAACAAACTTTTTTCAATTGACAGTTGCAGAATTGTGGA
127




 GT GG || TT|  ||A|TA AA|A A TTTTTT AATTGA|AGTTG|AGAATTGTGGA


Sbjct:
27972
CGTGGGACCCTTCAGCCACTACAACAGAATTTTTTAAATTGACAGTTGCAGAATTGTGGA
28033


Query:
  126
GTGTTTTTACATTGATCTTTTGCTAATGCAGTTAGCAGTATGTTTTGCATGTATGACTTA
67




GTGTTTTTA|ATTGAT|TTTTG|TAATG|A TTAG|A TATGTTTTG|ATGTATGA|TTA


Sbjct:
28032
GTGTTTTTACATTGATCTTTTGCTAATGCAATTAGCATTATGTTTTGCATGTATGACTTA
28091


Query:
   66
ATAAATCCTTGAATCATAAAAAAAAAAAAAAAAATGTCTTTGGAACTTGAAAAAAAA
10
(SEQ ID NO: 17)




ATAAAT||TTGAAT|ATA  A     AA A    TGT TTTG  A|TTGA  AA AA


Sbjct:
28092
ATAAATCCTTGAATCATACGACTGGTAATACTGGTGTTTTTGAGACTTGATGAACAA
28148
(SEQ ID NO: 18)






RISKMARKER2

RISKMARKER2 is a 650 bp rat expressed sequence tag (EST) [AW435096]. The nucleic acid sequence was initially identified in a cloned fragment having the following sequence:










  1
TTTTTTTTTTTTTTTTTGGCAGAATTCTGATGTTTACTGGGACCCATAGTAGTCAAGGTGACAGCAAGGGTAGGGGAGGA
   (SEQ ID NO: 3)



 81
AACTCAGCAGAGGCGGATCCCAGGTCTGGAGGGAAGCTGACAGCAGCCCAGTAAGCTGTGCCAGAAGGCTGTAACAGTAG


161
CGGAGCCAGTGACAGCGCCAGGCTGGGCTGGGTTCTCTCTGTGGGTGTGCACGGCAAAGCTGCGGCCTGTGGGCCCTGGG


241
GGGCCTGTCAGCTCCACATCCACCACATGCATGTCGGTGAGGCTAAGGTCAGCCACAAGCACCCCAATGACACGATCAAA


321
GCCTAGACTGGGAGCGGCCAGGGCAGCGGCTGCCATGGTGTTGGAGTTTCGGGGGGCCAAGGGGCAGAGCCCACGCACAG


401
GGCCCTCATAGAGCACTGTGCGGGGCCCACTACTATGTGCGGCAGCCAGGGGTCNCTCCAGCCGGAAGCCATCAGGATGT


481
GTGG






The cloned sequence was assembled into a contig resulting in the following consensus sequence:










  1
TTTTTTTTTTTTTTTTTGGCAGAATTCTGATGTTTACTGGGACCCATAGTAGTCAAGGTGACAGCAAGGGTAGGGGAGGA
    (SEQ ID NO: 4)



 81
AACTCAGCAGAGGCGGATCCCAGGTCTGGAGGGAAGCTGACAGCAGCCCAGTAAGCTGTGCCAGAAGGCTGTAACAGTAG


161
CGGAGCCAGTGACAGCGCCAGGCTGGGCTGGGTTCTCTCTGTGGGTGTGCACGGCAAAGCTGCGGCCTGTGGGCCCTGGG


241
GGGCCTGTCAGCTCCACATCCACCACATGCATGTCGGTGAGGCTAAGGTCAGCCACAAGCACCCCAATGACACGATCAAA


321
GCCTAGACTGGGAGCGGCCAGGGCAGCGGCTGCCATGGTGTTGGAGTTTCGGGGGGCCAAGGGGCAGAGCCCACGCACAG


401
GGCCCTCATAGAGCACTGTGCGGGGCCCACTACTATGTGCGGCAGCCAGGGGTCNCTCCAGCCGGAAGCCATCAGGATGT


481
GTGGCCATGGTGACTCGAAGGCTCTGGAGGCCTCCGGCTGCATCCAATCTGCTGATGTCTTCACAACCCCACAGGGCCCC


561
TCGGGCCACAAACACCGTGTGGCCCCAGTGGTTTGAAGCCTCCAGGAGCTGCCGCTCTGTGGTCTGGTCAGCGAGAGCTG


641
AGGGGGATCC






Blast-N Results:

>gb:GENBANK-ID:AW435096|acc:AW435096 UI-R-BJ0p-afy-e-10-0-UI.s1 UI-R-BJ0p Rattus norvegicus cDNA clone UI-R-BJ0p-afy-e-10-0-UI 3′, mRNA sequence—Rattus norvegicus, 484 bp (RNA).

    
    
        Length=484

Plus Strand HSPs:

    
    














Query:
  1
TTTTTTTTTTTTTTTTTGGCAGAATTCTGATGTTTACTGGGACCCATAGTAGTCAAGGTG
60






||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
  1
TTTTTTTTTTTTTTTTTGGCAGAATTCTGATGTTTACTGGGACCCATAGTAGTCAAGGTG
60


Query:
 61
ACAGCAAGGGTAGGGGAGGAAACTCAGCAGAGGCGGATCCCAGGTCTGGAGGGAAGCTGA
120




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
 61
ACAGCAAGGGTAGGGGAGGAAACTCAGCAGAGGCGGATCCCAGGTCTGGAGGGAAGCTGA
120


Query:
121
CAGCAGCCCAGTAAGCTGTGCCAGAAGGCTGTAACAGTAGCGGAGCCAGTGACAGCGCCA
180




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
121
CAGCAGCCCAGTAAGCTGTGCCAGAAGGCTGTAACAGTAGCGGAGCCAGTGACAGCGCCA
180


Query:
181
GGCTGGGCTGGGTTCTCTCTGTGGGTGTGCACGGCAAAGCTGCGGCCTGTGGGCCCTGGG
240




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
181
GGCTGGGCTGGGTTCTCTCTGTGGGTGTGCACGGCAAAGCTGCGGCCTGTGGGCCCTGGG
240


Query:
241
GGGCCTGTCAGCTCCACATCCACCACATGCATGTCGGTGAGGCTAAGGTCAGCCACAAGC
300




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
241
GGGCCTGTCAGCTCCACATCCACCACATGCATGTCGGTGAGGCTAAGGTCAGCCACAAGC
300


Query:
301
ACCCCAATGACACGATCAAAGCCTAGACTGGGAGCGGCCAGGGCAGCGGCTGCCATGGTG
360




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
301
ACCCCAATGACACGATCAAAGCCTAGACTGGGAGCGGCCAGGGCAGCGGCTGCCATGGTG
360


Query:
361
TTGGAGTTTCGGGGGGCCAAGGGGCAGAGCCCACGCACAGGGCCCTCATAGAGCACTGTG
420




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
361
TTGGAGTTTCGGGGGGCCAAGGGGCAGAGCCCACGCACAGGGCCCTCATAGAGCACTGTG
420


Query:
421
CGGGGCCCACTACTATGTGCGGCAGCCAGGGGTCCCTCCAGCCGGAAGCCATCAGGATGT
480




|||||||||||||||||||||||||||||||||| |||||||||||||||||||||||||


Sbjct:
421
CGGGGCCCACTACTATGTGCGGCAGCCAGGGGTCNCTCCAGCCGGAAGCCATCAGGATGT
480


Query:
421
GTGG
484
(SEQ ID NO: 19)




||||


Sbjct:
421
GTGG
484
(SEQ ID NO: 3)






Blast-X Results:

>ptnr:SPTREMBL-ACC:Q19527 F17C8.3 PROTEIN—Caenorhabditis elegans, 973 aa.

Top Previous Match Next Match

    
    
        Length=973

Minus Strand HSPs:

    
    










Score = 351 (123.6 bits), Expect = 6.3e-30, P = 6.3e-30



Identities = 78/161 (48%), Positives = 96/161 (59%), Frame = −1












Query:
650
GSPSALADQTTERQLLEASNHWGHTVFVARGALWGCEDISRLDAAGGLQSLRVTMATHPD
471






GSP+  A+Q    +L + S   G +  +  GALWG  DI  ++   G L+ L VTM HP


Sbjct:
530
GSPTCFANQELLEKLKLSLSHGKKLLIPAGALWGANDIQKMADVGSLKGLTVTMIKHPT
589


Query:
470
GFRLEGPLAAAHSSGP---RTVLYEGPVRGLCPLAPRNSNTMAAAALAAPSLGFDRVI
306




 F+L  PL +          TVLYEG VRGLCPLAP N NTMA  ALAA +LGFD V


Sbjct:
590
SFKLGSPLFEINEKAKLEETNETVLYEGSSVRGLCPLAPNNVNTMAGGALAASNLGFDEVK
649


Query:
305
GVLVADLSLTDMHVVDVELTGPPGPTGRSFAVHTHRENPAQPGAVT
168
(SEQ ID NO: 20)




  L++D  +TD HVV+V + G  G     F V T R NPA+PGAVT


Sbjct:
650
AKLISDPKMTDWHVVEVRVEGDDG-----FEVITRRNNPAKPGAVT
690
(SEQ ID NO: 21)






RISKMARER3
RISKMARKER3 is a 1019 nucleotide sequence encoding superoxide dismutase copper chaperone [AF255305]:










  1
ggtctctgga ccctaccggt tgtgtggccc aagcgggtga ctgcagccag gatggcttcg
      (SEQ ID NO: 22)



 61
aagtcggggg acggtggaac tatgtgtgcg ttggagttta cagtacagat gagttgtcag


121
agctgcgtgg acgctgtgca caagaccctg aaaggggcgg cgggtgtcca gaatgtggaa


181
gttcagttgg agaaccagat ggtgttggtg cagaccactt tgcccagcca ggaggtgcaa


241
gcgctcctgg aaagcacagg gaggcaggct gtactcaagg gcatgggcag cagccaacta


301
aagaatctgg gagcagcagt ggccattatg gagggcagtg gcaccgtaca gggggtggtc


361
cgcttcctac agctgtcctc tgagctctgc ctgattgagg gaaccatcga cggcctggag


421
cctgggctgc atgggcttca tgtccatcag tatggggacc ttaccaagga ctgcagcagc


481
tgtggggacc attttaaccc tgatggagca tctcatgggg gtcctcagga cactgatcgg


541
caccggggag atctgggcaa tgttcacgct gaagctagtg gccgagctac cttccggata


601
gaggataaac agctgaaggt gtgggatgtg attggccgca gtctggttgt tgatgaggga


661
gaagatgacc tgggccgggg aggccatccc ttatccaagg tcacagggaa ttctgggaag


721
aggttggcct gtggcatcat tgcacgctct gctggccttt tccagaatcc caagcagatc


781
tgctcctgtg atgggctcac tatctgggag gagcgaggcc ggcccattgc tggccaaggc


841
cgaaaggact cagcccaacc ccctgctcac ctctgaacag agcctcctgt caggttattc


901
agtcctccta gctgaacatc ttcctgcaga gggagcctca agcccttgct tgtataggcc


961
taaagggcag ataggcattg ttgtatcctg agcaaattaa attgttactc tcatatggc






RISKMARKER4

RISKMARKER4 is a 878 nucleotide sequence encoding alpha-2 microglubulin [U31287]:










  1
ggcacgagca gagagattgt cccaacagag aggcaattct attccctacc aacatgaagc
   (SEQ ID NO: 23)



 61
tgttgctgct gctgctgtgt ctgggcctga cactggtctg tggccatgca gaagaagcta


121
gttccacaag agggaacctc gatgtggcta agctcaatgg ggattggttt tctattgtcg


181
tggcctctaa caaaagagaa aagatagaag agaatggcag catgagagtt tttatgcagc


241
acatcgatgt cttggagaat tccttaggct tcaagttccg tattaaggaa aatggagagt


301
gcagggaact atatttggtt gcctacaaaa cgccagagga tggcgaatat tttgttgagt


361
atgacggagg gaatacattt actatactta agacagacta tgacagatat gtcatgtttc


421
atctcattaa tttcaagaac ggggaaacct tccagctgat ggtgctctac ggcagaacaa


481
aggatctgag ttcagacatc aaggaaaagt ttgcaaaact atgtgaggcg catggaatca


541
ctagggacaa tatcattgat ctaaccaaga ctgatcgctg tctccaggcc cgaggatgaa


601
gaaaggcctg agcctccagt gctgagtgga gacttctcac caggactcta gcatcaccat


661
ttcctgtcca tggagcatcc tgagacaaat tctgcgatct gatttccatc ctctgtcaca


721
gaaaagtgca atcctggtct ctccagcatc ttccctaggt tacccaggac aacacatcga


781
gaattaaaag ctttcttaaa tttctcttgg ccccacccat gatcattccg cacaaatatc


841
ttgctcttgc agttcaataa atgattaccc ttgcactt






RISKMARKER5

RISKMARKER5 is a 2443 bp rat mRNA for Mx3 protein [X52713]. The nucleic acid was initially identified in a cloned fragment (having 100% sequence identity to the rat mRNA) having the following sequence:










  1
CCATGGATGAAATCTTCCAGCATCTGAATGCCTACCGCCAGGAGGCTCACAACTGCATCTCCAGCCACATTCCATTGATC
   (SEQ ID NO: 5)



 81
ATCCAGTATTTCATCTTGAAGATGTTTGCTGAGAAGCTGCAGAAGGGCATGCTCCAGCTCCTGCAGGACAAGGATTCCTG


161
CAGCTGGCTCCTGAAGGAAAAGAGTGACACCAGTGAGAAGAGGAGATTCCTGAAGGAGCGGTTGGCAAGGCTGGCCCAAG


241
CTCAGCGCAGGCTAGC






Blast-N Results:

>gb:GENBANK-ID:RNMX3|acc:X52713 Rat mRNA for Mx3 protein—Rattus norvegicus, 2443 bp.

Top Previous Match Next Match

    
    
        Length=2443

Plus Strand MSPs:

    
    










Score = 1280 (192.1 bits), Expect = 9.5e-52, P = 9.5e-52



Identities = 256/256 (100%), Positives = 256/256 (100%), Strand = Plus/Plus












Query:
   1
CCATGGATGAAATCTTCCAGCATCTGAATGCCTACCGCCAGGAGGCTCACAACTGCATCT
60
 (SEQ ID NO: 24)





||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
1710
CCATGGATGAAATCTTCCAGCATCTGAATGCCTACCGCCAGGAGGCTCACAACTGCATCT
1769


Query:
  61
CCAGCCACATTCCATTGATCATCCAGTATTTCATCTTGAAGATGTTTGCTGAGAAGCTGC
120




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
1770
CCAGCCACATTCCATTGATCATCCAGTATTTCATCTTGAAGATGTTTGCTGAGAAGCTGC
1829


Query:
 121
AGAAGGGCATGCTCCAGCTCCTGCAGGACAAGGATTCCTGCAGCTGGCTCCTGAAGGAAA
180




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
1830
AGAAGGGCATGCTCCAGCTCCTGCAGGACAAGGATTCCTGCAGCTGGCTCCTGAAGGAAA
1889


Query:
 181
AGAGTGACACCAGTGAGAAGAGGAGATTCCTGAAGGAGCGGTTGGCAAGGCTGGCCCAAG
240




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
1890
AGAGTGACACCAGTGAGAAGAGGAGATTCCTGAAGGAGCGGTTGGCAAGGCTGGCCCAAG
1949


Query:
 241
CTCAGCGCAGGCTAGC
256
(SEQ ID NO: 5)




||||||||||||||||


Sbjct:
1950
CTCAGCGCAGGCTAGC
1965






Blast-X Results:

>ptnr:SWISSPROT-ACC:P18590 INTERFERON-INDUCED GTP-BINDING PROTEIN MX3—Rattus norvegicus (Rat), 659 aa.

Top Previous Match Next Match

    
    
        Length=659

Plus Strand HSPs:

    
    










Score = 429 (151.0 bits), Expect = 5.3e-39, P = 5.3e-39



Identities = 84/84 (100%), Positives = 84/84 (100%), Strand = +3












Query:
  3
MDEIFQHLNAYRQEAHNCISSHIPLIIQYFILKMFAEKLQKGMLQLLQDKDSCSWLLKEK
182






MDEIFQHLNAYRQEAHNCISSHIPLIIQYFILKMFAEKLQKGMLQLLQDKDSCSWLLKEK


Sbjct:
571
MDEIFQHLNAYRQEAHNCISSHIPLIIQYFILKMFAEKLQKGMLQLLQDKDSCSWLLKEK
630


Query:
183
SDTSEKRRFLKERLARLAQAQRRL
254
(SEQ ID NO: 25)




SDTSEKRRFLKERLARLAQAQRRL


Sbjct:
631
SDTSEKRRFLKERLARLAQAQRRL
654
(SEQ ID NO: 26)






RISKMARKER6

RISKMARKER6 is 369 bp novel gene fragment, which has 98% amino acid identity (90% nucleic acid sequence identity) to Human ERj3 protein [AJ250137]. The nucleic acid sequence was initially identified in a cloned fragment having the following sequence:










  1
TCTAGAAAGTCACCTTGGAAGAAGTGTACGCAGGGAACTTTGTGGAAGTAGTTAGAAACAAGCCCGTAGCCAGGCAGGCT
(SEQ ID NO: 6)



 81
CCTGGCAAACGGAAATGCAACTGTCGGCAGGAGATGCGAACCACACAGCTGGGACCAGGGCGCTTCCAAATGACCCAGGA


161
AGTGGTTTGTGACGAGTGCCCTAATGTCAAACTAGTGAATGAAGAACGAACACTAGAAGTGGAAATAGAGCCTGGGGTGA


241
GAGATGGCATGGAGTACCCCTTTATTGGAGAAGGTGAGCCTCATGTGGATGGGGAACCCGGAGACTTACGGTTCCGAATC


321
AAAGTTGTCAAGCACCGGATATTTGAGAGGAGAGGGGATGACCTGTACA






Blast-N Results: >gb:GENBANK-ID:HSA2501371|acc:AJ250137 Homo sapiens mRNA for ERj3 protein (ERj3 gene)—Homo sapiens, 1159 bp.

Top Previous Match Next Match

    
    
        Length , 1159

Plus Strand HSPs:

    
    










Score = 1524 (228.7 bits), Expect = 5.6e-63, P = 5.6e-63



Identities = 334/369 (90%), Positives = 334/369 (90%), Strand = Plus/Plus












Query:
  1
TCTAGAAAGTCACCTTGGAAGAAGTGTACGCAGGGAACTTTGTGGAAGTAGTTAGAAACA
60






TCTAGAA GTCAC TTGGAAGAAGT TA GCAGG AA TTTGTGGAAGTAGTTAGAAACA


Sbjct:
431
TCTAGAA-GTCACTTTGGAAGAAGTATATGCAGGAAATTTTGTGGAAGTAGTTAGAAACA
489


Query:
 61
AGCCCGTAGCCAGGCAGGCTCCTGGCAAACGGAAATGCAACTGTCGGCAGGAGATGCGAA
120




A CC GT GC AGGCAGGCTCCTGGCAAACGGAA TGCAA TGTCGGCA GAGATGCG A


Sbjct:
490
AACCTGTGGCAAGGCAGGCTCCTGGCAAACGGAAGTGCAATTGTCGGCAAGAGATGCGGA
549


Query:
121
CCACACAGCTGGGACCAGGGCGCTTCCAAATGACCCAGGAAGTGGTTTGTGACGAGTGCC
180




CCAC CAGCTGGG CC GGGCGCTTCCAAATGACCCAGGA GTGGT TG GACGA TGCC


Sbjct:
550
CCACCCAGCTGGGCCCTGGGCGCTTCCAAATGACCCAGGAGGTGGTCTGCGACGAATGCC
609


Query:
181
CTAATGTCAAACTAGTGAATGAAGAACGAACACTAGAAGTGGAAATAGAGCCTGGGGTGA
240




CTAATGTCAAACTAGTGAATGAAGAACGAAC CT GAAGT GAAATAGAGCCTGGGGTGA


Sbjct:
610
CTAATGTCAAACTAGTGAATGAAGAACGAACACTAGAAGTGGAAATAGAGCCTGGGGTGA
669


Query:
241
GAGATGGCATGGAGTACCCCTTTATTGGAGAAGGTGAGCCTCATGTGGATGGGGAACCCG
300




GAGA GGCATGGAGTACCCCTTTATTGGAGAAGGTGAGCCTCA GTGGATGGGGA CC G


Sbjct:
670
GAGATGGCATGGAGTACCCCTTTATTGGAGAAGGTGAGCCTCATGTGGATGGGGAACCCG
729


Query:
301
GAGACTTACGGTTCCGAATCAAAGTTGTCAAGCACCGGATATTTGAGAGGAGAGGGGATG
360




GAGA TTACGGTTCCGAATCAAAGTTGTCAAGCACC  ATATTTGA AGGAGAGG GATG


Sbjct:
730
GAGACTTACGGTTCCGAATCAAAGTTGTCAAGCACCGGATATTTGAGAGGAGAGGGGATG
789


Query:
361
ACCTGTACA
369
(SEQ ID NO: 6)




A  TGTACA


Sbjct:
790
ATTTGTACA
798
(SEQ ID NO: 27)






Blast-X Results:

>ptnr:SPTREBML-ACC:Q9UBS4 ERJ3 PROTEIN PRECURSOR—Homo sapiens (Human), 358 aa.

Top Previous Match Next Match

    
    
        Length=358

Plus Strand HSPs:

    
    










Score = (224.2 bits), Expect = 2.1e-61, P = 2.1e-61



Identities = 119/121 (98%), Positives = 120/121 (99%), Frame = +3












Query:
  6
KVTLEEVYAGNFVEVVRNKPVARQAPGKRKCNCRQEMRTTQLGPGRFQMTQEVVCDECPN
185






+VTLEEVYAGNFVEVVRNKPVARQAPGKRKCNCRQEMRTTQLGPGRFQMTQEVVCDECPN


Sbjct:
139
EVTLEEVYAGNFVEVVRNKPVARQAPGKRKCNCRQEMRTTQLGPGRFQMTQEVVCDECPN
198


Query:
186
VKLVNEERTLEVEIEPGVRDGMEYPFIGEGEPHVDGEPGDLRFRIKVVKHRIFERRGDDL
365




VKLVNEERTLEVEIEPGVRDGMEYPFIGEGEPHVDGEPGDLRFRIKVVKH IFERRGDDL


Sbjct:
139
VKLVNEERTLEVEIEPGVRDGMEYPFIGEGEPHVDGEPGDLRFRIKVVKHPIFERRGDDL
258


Query:
366
Y
368
(SEQ ID NO: 28)




Y


Sbjct:
259
Y
259
(SEQ ID NO: 29)






RISKMARKER7

RISKMARKER7 is a 594 bp novel gene fragment, which has 65% sequence identity to Mus musculus hexokinase II [AJ238540], probable 3′UTR. The nucleic acid sequence was initially identified in a cloned fragment having the following sequence:










  1
GGGCCCCACTAAAACATACACAAAAGAATAAAAATGTTCATTTTAAACTTAAACTGCTTCCTGGTTTTACAAGGCATAAA
   (SEQ ID NO: 7)



 81
TATATAGCATCTCCAACAGCTACCTGTAGATTCTGTTAGTGCAAAACCTTAGAAACCCTCCTGGAGCTCAAAGGCATCCG


161
GACTAGT






The cloned sequence was assembled into a contig resulting in the following consensus sequence:










  1
TTTTTTTTTTTTTTTTTAAAAAAGATTATAAAATTGAATTTATTGAGTTTCACACAAGATGCACTTATAAAATTAGTACT
   (SEQ ID NO: 8)



 81
GAATGCCATTATGACAGAAGTGAGCATCATCCACACTCCCAAGAGCATCTGCAAAGGAAATCAATCTTCAGAGAATAGCA


161
CAGAAACAGAAAATCCAAGCGAACAAAAAGATACATCTAGGCCGTGTTCTTGTTCTGACCAGGGCCGCATTTGGCAAAGC


241
TTTCTCTGCACCTCCCCTGGTTGCCAAGGATACTTTCTTTTGTTAAAAAAAAAAGTTTAGAAGTGGGGCCCCACTAAAAC


321
ATACACAAAAGAATAAAAATGTTCATTTTAAACTTAAACTGCTTCCTGGTTTTACAAGGCATAAATATATAGCATCTCCA


401
ACAGCTACCTGTAGATTCTGTTAGTGCAAAACCTTAGAAACCCTCCTGGAGCTCAAAGGCATCCGGACTAGTTTTGTACT


481
TAAACAGGATACGGGTAAACCACTTAAAATTTGCCATCTCTGCCCAAAGTGTTTGCATGAGAACTGAGTTTCAGAAGACA


561
GCATAGGAAAGAGTCAGAAACGGTCAACTTTTTT






Blast-N Results:

>gb:GENBANK-ID:MMU238540|acc:AJ238540 Mus musculusmRNA for hexokinase II—Mus musculus, 5474 bp.
Top Previous Match Next Match

    
    
        Length=5474

Minus Strand HSPs:

    
    










Score = 251 (37.7 bits), Expect = 0.045, P = 0.044



Identities = 121/184 (65%), Positives = 121/184 (65%), Strand = Minus/Plus












Query:
 184
GTTCGCTTGGATTTT-CTG-TTTCTGTGCTATTCTCTGAAGATT-GATTTCCTTTGCAGA
128






G TC CT  G T  T CTG TTT TGTG T TTC  TGAA  TT GA T C T T CA A


Sbjct:
5287
GCTCTCTCTGCTAATGCTGCTTTGTGTGATCTTCAGTGAACCTTTGACT-CATCT-CATA
5344


Query:
 127
TGCTCTTGGGAGTGTGGATGATGCTCACTTCTGTCATAATGG-CATTC-AGTACTAATTT
70




T C CT GG A T  G  T  TG  C  TT TGTCAT ATG  CA T  AG ACTA TT


Sbjct:
5345
TCC-CTGGGCACTCGGTCTAGTGAGCGTTT-TGTCATCATGTACAGTAGAGAACTAGTTG
5402


Query:
  69
TATAAGTGCATCTTGTGTGAAACTCA-ATAAATTCAATTTTATAATCTTTTTTAAAAAAA
11




 AT A   CAT T  TGT AA CT   AT AAT  AATTTTA   T TTTTTT AAAAAA


Sbjct:
5403
AATTAAC-CATGTGATGTTAA-CTATTATTAATA-AATTTTAACTTTTTTTTTCAAAAAA
5459


Query:
  10
AAAAAAAAAA
1
(SEQ ID NO: 30)




AAAAAAAAAA


Sbjct:
5460
AAAAAAAAAA
5469
(SEQ ID NO: 31)








Score = 250 (37.5 bits), Expect = 0.051, P = 0.049



Identities = 122/184 (66%), Positives = 122/184 (66%), Strand = Minus/Plus












Query:
 184
GTTCGCTTGGATTTT-CTG-TTTCTGTGCTATTCTCTGAAGATT-GATTTCCTTTGCAGA
128






G TC CT  G T  T CTG TTT TGTG T TTC  TGAA  TT GA T C T T CA A


Sbjct:
5287
GCTCTCTCTGCTAATGCTGCTTTGTGTGATCTTCAGTGAACCTTTGACT-CATCT-CATA
5344


Query:
 127
TGCTCTTGGGAGTGTGGATGATGCTCACTTCTGTCATAATGG-CATTC-AGTACTAATTT
70




T C CT GG A T  G  T  TG  C  TT TGTCAT ATG  CA T  AG ACTA TT


Sbjct:
5345
TCC-CTGGGCACTCGGTCTAGTGAGCGTTT-TGTCATCATGTACAGTAGAGAACTAGTTG
5402


Query:
  69
TATAAGTGCATCTTGTGTGAAACTCA-ATAAATTCAATTTTATAATCTTTTTT-AAAAAA
12




 AT A   CAT T  TGT AA CT   AT AAT  AATTTTA   T TTTTTT AAAAAA


Sbjct:
5403
AATTAAC-CATGTGATGTTAA-CTATTATTAATA-AATTTTAACTTTTTTTTTCAAAAAA
5459


Query:
  11
AAAAAAAAAA
1
(SEQ ID NO: 30)




AAAAAAAAAA


Sbjct:
5460
AAAAAAAAAA
5469
(SEQ ID NO: 32)






Blast-X Results:

>ptnr:SPTREMBL-ACC:Q9VIA2 MST84DB PROTEIN—Drosophila melanogaster (Fruit fly), 70 aa.

Top Previous Match Next Match

    
    
        Length=70

Plus Strand HSPs:

    
    










Score = 66 (23.2 bits), Expect = 2.2, P = 0.88



Identities = 15/48 (31%), Positives = 25/48 (52%), Frame = +3












Query:
66
YKISTECHYDRSEHHPHSQEHLQRKS------IFRE*HRNRKSKRTKR
191
(SEQ ID NO:33)





YK+ ++ H  R +H P S++   RK        I ++  RNRK  R ++


Sbjct:
 3
YKVHSKVHKARMDHSPRSKDRKDRKGRKAHSKIHKDYSRNRKDHRVRK
50
(SEQ ID NO:34)






RISKMARKER8

RISKMARKER8 is a 797 bp novel gene fragment, which has 94% amino acid identity (79% nucleic acid sequence identity) to human GT335 mRNA (ES1 Protein Homolog) [U53003]. The nucleic acid was initially identified in a cloned fragment having the following sequence:










  1
CCTAGGACTGCACAACGTGAGTCCTTGAACCAGGCTCTGGAAAAGGTGCCCAGACCACCCAATGGGGACACACAGTGAGG
(SEQ ID NO:9)



 81
CCAGCCCCCAGTGAAATTCCTGCTGCTACCTGGGGCCCTTGGTGAGACTCTGGCTTCCGGCTGGTAGAAGCCAAGGTTGG


161
ACGCATAGTTGCAAAGCTCCTCCTTCAGGCACAAAGTGTCTATGCTTCTAATAGAACAGCAGCTCCCGTGTCCTGGCTGA


241
CAGCACCTTCTTCACCATGG






The cloned sequence was assembled into a contig resulting in the following consensus sequence:










  1
TTTTTTTTTTTTTTTTTTTTTTGAGTTTCCACTGTGGAAAAGAGTTTATTGTATGGCTGCAGGGATCTACTACAGAATCC
(SEQ ID NO:10)



 81
CCCTGGCTGCAGTTAGCTGTGCTTACTCTGGACATATCTCCGAAGACTTGGAGCCTAAATGGTTTTCTTCTTTTAGAGCT


161
TTAGTACCCGATCCATCAGACCTAGGACTGCACAACGTGAGTCCTTGAACCAGGCTCTGGAAAAGGTGCCCAGACCACCC


241
AATGGGGACACACAGTGAGGCCAGCCCCCAGTGAAATTCCTGCTGCTACCTGGGGCCCTTGGTGAGACTCTGGCTTCCGG


321
CTGGTAGAAGCCAAGGTTGGACGCATAGTTGCAAAGCTCCTCCTTCAGGCACAAAGTGTCTATGCTTCTAATAGAACAGC


401
AGCTCCCGTGTCCTGGCTGACCGGAGCACACAGGCTGAGCGTGCCACAGCGACGACGGAGGCCAAGCGTGGTGCTGGTGG


481
TGTTACTTTCCCGTGAGTTCCAGCACCTTCTTCACCATGGCCCCAATCCCGTCGTGGATGTGGTGGAGTTCGGTCTCACA


561
CATGAAGGCCGGGGTGGTGACCACCTTGTTTTTCTGGTCGACGTGAGCTTCGGTCACACCCTTCACACAGTGCTTGGCAC


641
CCAGGGCTTTGACGGCTTCCGCGGTTCCAGCATATGGCCACTTGCCCCCCTCCTCTTGCTCATGGCCCACGGTGACCTCC


721
ACACCTTTGATCACTTTGGCTGCGAGGACAGGAGCGATGCAGCATAGGCCAATGGGCTTCTTGGCTCCGTGGAATTC






Blast-N Results:

>gb:GENBANK-ID:HSU53003|acc:U53003 Human GT335 mRNA, complete cds—Homo sapiens, 1652 bp.

Top Previous Match Next Match

    
    
        Length=1652

Minus Strand HSPs:

    
    










Score = 1141 (171.2 bits), Expect = 7.9e-46, p = 7.9e-46



Identities = 307/385 (79%), Positives = 307/385 (79%), Strand = Minus/Plus












Query:
797
GAATTCCACGGAGCCAAGAAGCCCATTGGCCTATGCTGCATCGCTCCTGTCCTCGCAGCC
738
(SEQ ID NO:35)





GA TTCCAC   GCC  GAAGCCCAT GGC T TGCTGCAT GC CCTGTCCTCGC GCC


Sbjct:
577
GAGTTCCACCAGGCCGGGAAGCCCATCGGCTTGTGCTGCATTGCACCTGTCCTCGCGGCC
636
(SEQ ID NO:36)


Query:
737
AAAGTGATCAAAGGTGTGGAGGTCACCGTGGGCCATGAGCAAGAGGAGGGGGGCAAGTGG
678




AA GTG TCA AGG GT GAGGT AC GTGGGCCA GAGCA GAGGA GG GGCAAGTGG


Sbjct:
637
AAGGTGCTCAGAGGCGTCGAGGTGACTGTGGGCCACGAGCAGGAGGAAGGTGGCAAGTGG
696


Query:
677
CCATATGCTGGAACCGCGGAAGCCGTCAAAGCCCTGGGTGCCAAGCACTGTGTGAAGGGT
618




CC TATGC GG ACCGC GA GCC TCAA GCCCTGGGTGCCAAGCACTG GTGAAGG


Sbjct:
697
CCTTATGCCGGGACCGCAGAGGCCATCAAGGCCCTGGGTGCCAAGCACTGCGTGAAGGAA
756


Query:
617
GTGACCGAAGCTCACGTCGACCAGAAAAACAAGGTGGTCACCACCCCGGCCTTCATGTGT
558




GTG  CGAAGCTCACGT GACCAGAAAAACAAGGTGGTCAC ACCCC GCCTTCATGTG


Sbjct:
757
GTGGTCGAAGCTCACGTGGACCAGAAAAACAAGGTGGTCACGACCCCAGCCTTCATGTGC
816


Query:
557
GAGACCGAACTCCACCACATCCACGACGGGATTGGGGCCATGGTGAAGAAGGTGCTGGAA
498




GAGAC G ACTCCAC ACATCCA GA GGGAT GG GCCATGGTGA GAAGGTGCTGGAA


Sbjct:
817
GAGACGGCACTCCACTACATCCATGATGGGATCGGAGCCATGGTGAGGAAGGTGCTGGAA
876


Query:
497
CTCACGGGAAAGTAACAC-CACC-A-GCACCAC-GCTTGGCCTCCGT-CGTCGCTGTGGC
443




CTCAC GGAAAGT AC C CA   A G   C C GCT GGC  C G  C T GC  T  C


Sbjct:
877
CTCACTGGAAAGTGACGCGCATGGACGGGGCCCAGCTAGGCGCCAGGACTTGGCC-T--C
933


Query:
442
ACGCTCAGCCTGTGT-GCTC-CGGTCAGC
416




AC CTC G CTG G  GCT  CGG C GC


Sbjct:
934
ACCCTCTGGCTGAGGAGCTGTCGG-CTGC
961






Blast-X Results:

>ptnr:SWISSNEW-ACC:P30042 ES1 PROTEIN HOMOLOG, MITOCRONDRIAL PRECURSOR (PROTEIN KNP-I) (GT335 PROTEIN)—Homo sapiens (Human) 268 aa.

Top Previous Match Next Match

    
    
        Length=268

Minus Strand HSPs:

    
    










Score = 505 (177.8 bits), Except = 2.0e-47, P = 2.0e-47



Identities = 94/104 (90%), Positives = 99/104 (95%), Frame = -1












Query:
797
EFHGAKKPIGLCCIAPVLAAKVIKGVEVTVGHEQEEGGKWPYAGTAEAVKALGAKHCVKG
618
(SEQ ID NO:37)





EFH A KPIGLCCIAPVLAAKV++GVEVTVGHEQEEGGKWPYAGTAEA+KALGAKHCVK


Sbjct:
165
EFHQAGKPIGLCCIAPVLAAKVLRGVEVTVGHEQEEGGKWPYAGTAEAIKALGAKHCVKE
224
(SEQ ID NO:38)


Query:
617
VTEAHVDQKNKVVTTPAFMCETELHHIHDGIGAMVKKVLELTGK
486




V EAHVDQKNKVVTTPAFMCET LH+IHDGIGAMV+KVLELTGK


Sbjct:
225
VVEAHVDQKNKVVTTPAFMCETALHYIHDGIGAMVRKVLELTGK
268






Principle components analysis was used to generate three eigenvectors used to transform the original expression level data matrix, as shown in Table 4 below. Eigenvector 1 values represent NSAIDs with low risk of hepatoxicity, Eigenvector 2 values represent NSAIDs with very low risk of hepatoxicity, and Eigenvector 3 values represent NSAIDs with overdose risk of hepatoxicity.







TABLE 4







Transform Eigenvectors for Hepatoxicity


Markers by Risk Classification










Gene
Eigenvector 1
Eigenvector 2
Eigenvector 3













RISKMARKER 1
26.9
6.7
−0.9


RISKMARKER 2
23.3
−1.4
1.5


RISKMARKER 3
−26.0
−1.5
−2.3


RISKMARKER 4
12.6
−2.2
−6.4


RISKMARKER 5
18.0
−1.3
−3.1


RISKMARKER 6
−13.8
4.71
19.3


RISKMARKER 7
−29.7
−7.5
1.3


RISKMARKER 8
19.3
1.2
−2.6


% of variation
99.6
0.4
0.1


explained









These eigenvectors may be used to transform the expression levels of RISKMARKERS 1-8 (“RISKMARKERS”) in response to a given drug, in order to determine that drug's hepatotoxicity risk. For example, expression levels of RISKMARKERS correlating with Eigenvector 1 indicates that the test drug has a low risk of hepatotoxicity. Alternatively, a drug's RISKMARKERS expression profile can be generated simultaneously with the above-described training set (or an equivalent set) run in parallel with the test drug, and expression levels associated with the test drug directly compared to those of the training set.
A second training set based on type of injury (hepatocellular damage, cholestasis, elevated transaminase level) was also constructed, utilizing the compounds indicated in Table 5, below.







TABLE 5







Training Set of NSAIDs by Injury Type















Elevated



Control
Hepatocellular
Cholestasis
transmainases







Sterile water
Acetaminophen
Benoxaprofen
Zomepirac



10% ethanol
Flurbiprofen
Nabumetone
Mefenamic acid



Canola oil
Ketoprofen
Sulindac
Tenoxicam










This analysis produced ten fragments that significantly (p=8.7×10−18) discriminated among the drugs in the test set. The identities of these ten fragments (INJURYMARKER 1-10) that are included in the discriminatory set (with GenBank accession numbers) are shown below. Where appropriate, the cloned sequence from isolation is provided, and this sequence was then extended using either Genbank rat ESTs or from internally sequenced (Curagen Corporation) rat fragments. The fragments were used to extend the cloned sequence, and the extended contig sequence is provided as “consensus.” Finally, the best BlastN and BlastX results are also provided. In some instances the cloned sequence is identical to a known rat gene, in those instances the name of the gene and the GenBank accession number is provided.
INJURYMARKER 1
INJURYMARKER1 is a 1025 bp rat express sequence tag (EST) [AI169175]. The nucleic acid was initially identified in a cloned fragment having the following sequence:










  1
CTGATTTCAAATTTTTATTAGAGAACACTTTCGGATTTCAAATTTTTATTACAGAACAAACATTTTCTGATTTCAAATTT
(SEQ ID NO:11)



 81
CTATTATAATTCTCCAGTAATCAAAGCAGTGGCGTTGGCATGAAGGCAGACAGAGGTCATGGAAGAGACCAGGCTCAGAA


161
ACAGCCCCACCATGCACAGCGGGATGTTTTCCCACCAAGGGCAACATGCAAAGCCAGGTATCCACATGGGTAGAGTAGAA


241
AGTCAGACCTTACATCTCACACACAAATGAACTCAAAATATACCAGAGAGCAAAGCTAAGAGCTAAAATCAAGTTTCCTA


321
GGGCAAGCTGTAGTAGGTCCCTTGGGTGGGTTAATGCTTTTGTGGATGTGACTACCAAAAATTCAAACCAGAGCCAACGA


401
CCCAACTATTAATGGGCAGTGGACCTAAAGAGATTTCTTCAAACGATATATAAAGAAGGCCACCAAGCATATAAAACATG


481
TGACATCAGTAGTCAGAGAGATGGGAAGCAGAAGCACTAGCAGATCTTAACACCTACTAGAACANCCACTAAAAAAGAGT


561
AAGACTCACAAGGACATGGGCACTTCTAATCTCTGTGCACTGCTGCCAGGACATACAATAGTGTGGTCACTATGGAGACT


641
ACGGCAGTGCCTACTAATAACAGCAGAGTTACCCTAAGACATACAATCTGCTGCGTGTATGCTAAGCAGGATCCGAGGGA


721
TATTTGTATATACATGTTCACAGCATAGTCAGGAGCTCCAGGGTGGGAACAACTGAGGTACC






The cloned sequence was assembled into a contig resulting in the following consensus sequence:










  1
CTGATTTCAAATTTTTATTATAGAACACTTTCTGATTTCAAATTTTTATTACAGAACAAACATTTTCTGATTTCAAATTT
(SEQ ID NO:12)



 81
CTATTATAATTCTCCAGTAATCAAAGCAGTGGCGTTGGCATGAAGGCAGACAGAGGTCATGGAAGAGACCAGGCTCAGAA


161
ACAGCCCCACCATGCACAGCGGGATGTTTTCCCACCAAGGGCAACATGCAAAGCCAGGTATCCACATGGGTAGAGTAGAA


241
AGTCAGACCTTACATCTCACACACAAATGAACTCAAAATATACCAGAGAGCAAAGCTAAGAGCTAAAATCAAGTTTCCTA


321
GGGCAAGCTGTAGTAGGCTCCCTTGGGTGGGTTAATGCTTTTGTGGATGTGACTACCAAAAATTCAACCAGAGCCAACGA


401
CCCAACTATTAATGGGCAGTGGACCTAAAGAGATTTCTTCAAACGATATATAAAGAAGGCCACCAAGCATATAAAACATG


481
TGACATCAGTAGTCAGAGAGATGGGAAGCAGAAGCACTAGCAGATCTTAACACCTACTAGAACAGCCACTAAAAAAGAGT


561
AAGACTCACAAGGACATGGGCACTTCTAATCTCTGTGCACTGCTGCCAGGACATACAATAGTGTGGTCACTATGGAGACT


641
ACGGCAGTGCCTACTAATAACAGCAGAGTTACCCTAAGACATACAATCTGCTGCGTGTATGCTAAGCAGGATCCGAGGGA


721
TATTTGTATATACATGTTCACAGCATAGTCAGGAGCTCCAGGGTGGGAACAACTGAGGTACCCACGGCTGGATGAGTAGG


801
TAACAAGAAACATACAGCATACATACAACACACACTAAAGTCTAAAGTACTATTTGTCCTTACAAAGGAAACTCATACAT


881
GATACAAGCCTTCACGGCATTCTGCTACATGAACACGCACACACACACACACACACACACACACACACGCACTGAGAATC


961
TATGTATACCAGGCACTTAGGGTACTCAAATTCAGAAACAGGACAGAGAATGGTGATTGCCATGG






Blast-N Results:

>gb:GENBANK-ID:AI169175|acc:AI169175 EST215009 Normalized rat kidney, Bento Soares Rattus sp. CDNA clone RKIBO44 3′ end, mRNA sequence—Rattus sp., 670 bp (RNA).

Top Previous Match Next Match

    
    
        Length=670

Plus Strand HSPs:

Score=3305 (495.9 bits), Expect=4.3e-143, P=4.3e-143 Identities=661/661 (100%), Positives=661/661 (100%), Strand=Plus/Plus

    
    










Score = 3305 (495.9 bits), Except = 4.3e-143, P = 4.3e-143



Identities = 661/661 (100%), Positives = 661/661 (100%), Strand = Plus/Plus












Query:
  4
ATTTCAAATTTTTATTATAGAACACTTTCTGATTTCAAATTTTTATTACAGAACAAACAT
63
(SEQ ID NO:39)





||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
  1
ATTTCAAATTTTTATTATAGAACACTTTCTGATTTCAAATTTTTATTACAGAACAAACAT
60
(SEQ ID NO:40)


Query:
 64
TTTCTGATTTCAAATTTCTATTATAATTCTCCAGTAATCAAAGCAGTGGCGTTGGCATGA
123




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
 61
TTTCTGATTTCAAATTTCTATTATAATTCTCCAGTAATCAAAGCAGTGGCGTTGGCATGA
120


Query:
124
AGGCAGACAGAGGTCATGGAAGAGACCAGGCTCAGAAACAGCCCCACCATGCACAGCGGG
183




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
121
AGGCAGACAGAGGTCATGGAAGAGACCAGGCTCAGAAACAGCCCCACCATGCACAGCGGG
180


Query:
184
ATGTTTTCCCACCAAGGGCAACATGCAAAGCCAGGTATCCACATGGGTAGAGTAGAAAGT
243




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
181
ATGTTTTCCCACCAAGGGCAACATGCAAAGCCAGGTATCCACATGGGTAGAGTAGAAAGT
240


Query:
244
CAGACCTTACATCTCACACACAAATGAACTCAAAATATACCAGAGAGCAAAGCTAAGAGC
303




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
241
CAGACCTTACATCTCACACACAAATGAACTCAAAATATACCAGAGAGCAAAGCTAAGAGC
300


Query:
304
TAAAATCAAGTTTCCTAGGGCAAGCTGTAGTAGGCTCCCTTGGGTGGGTTAATGCTTTTG
363




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
301
TAAAATCAAGTTTCCTAGGGCAAGCTGTAGTAGGCTCCCTTGGGTGGGTTAATGCTTTTG
360


Query:
364
TGGATGTGACTACCAAAAATTCAACCAGAGCCAACGACCCAACTATTAATGGGCAGTGGA
423




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
361
TGGATGTGACTACCAAAAATTCAACCAGAGCCAACGACCCAACTATTAATGGGCAGTGGA
420


Query:
424
CCTAAAGAGATTTCTTCAAACGATATATAAAGAAGGCCACCAAGCATATAAAACATGTGA
483




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
421
CCTAAAGAGATTTCTTCAAACGATATATAAAGAAGGCCACCAAGCATATAAAACATGTGA
480


Query:
484
CATCAGTAGTCAGAGAGATGGGAAGCAGAAGCACTAGCAGATCTTAACACCTACTAGAAC
543




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
481
CATCAGTAGTCAGAGAGATGGGAAGCAGAAGCACTAGCAGATCTTAACACCTACTAGAAC
540


Query:
544
AGCCACTAAAAAAGAGTAAGACTCACAAGGACATGGGCACTTCTAATCTCTGTGCACTGC
603




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
541
AGCCACTAAAAAAGAGTAAGACTCACAAGGACATGGGCACTTCTAATCTCTGTGCACTGC
600


Query:
604
TGCCAGGACATACAATAGTGTGGTCACTATGGAGACTACGGCAGTGCCTACTAATAACAG
663




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
601
TGCCAGGACATACAATAGTGTGGTCACTATGGAGACTACGGCAGTGCCTACTAATAACAG
660


Query:
664
C
664




|


Sbjct:
661
C
661






INJURYMARKER2
INJURYMARKER2 is a 893 nucleotide sequence encoding phosphotidylethanolamine N-methyltransferase [L14441]:










  1
tccccgctga gttcatcacc agggacaggt gacctgagct gcccctggag cccagctccc
(SEQ ID NO:41)



 61
atttccttct ggttctggcc gatctcttcg ttatgagctg gctgctgggt tacgtggacc


121
ccacagagcc cagctttgtg gcggctgtgc tcaccattgt gttcaatcca ctcttctgga


181
atgtggtagc aaggtgggag cagagaactc gcaagctgag cagagccttc gggtcccctt


241
acctagcctg ctattccctg ggcagcatca tcctgcttct gaacatcctc cgctcccact


301
gcttcacaca ggccatgatg agccagccca agatggaggg cctggatagc cacaccatct


361
acttcctggg ccttgcactc ctgggctggg gactcgtgtt tgtgctctcc agcttctatg


421
cactggggtt cactgggacc tttctaggtg actactttgg gatcctcaag gagtccagag


481
tgaccacatt tcccttcagc gtgctggaca accccatgta ctggggaagt acagccaact


541
acctaggctg ggcacttatg cacgccagcc ctacaggcct gctgttgacg gtgctggtgg


601
cactcgtcta cgtggttgct ctcctgtttg aagagccctt cactgcggag atctaccggc


661
ggaaagccac caggttgcac aaaaggagct gacagggcca tgagggacct ttggaaagcc


721
ggattgcctc ccggctgacc caagcaacaa cccttctcgg ggagagcagc gctggccatt


781
gtacctgtgc cttggaaacc agtcatgggg gtgctcaggc attatgtcat gtgactgctg


841
agacccccat ccccaccaat ccctgacaca ataataaagg ctttgtgacc tcc






INJURYMARKER3
INJURYMARKER3 is a 1131 nucleotide hexokinase-encoding sequence [M86235]:










   1
agcaggaatc ccctccgctt gcgggtagga agcttgggga gcagcctcat ggaagagaag
(SEQ ID NO:42)



  61
cagatcctgt gcgtggggct ggtggtgctg gacatcatca atgtggtgga caaataccca


 121
gaggaagaca cggatcgcag gtgcctatcc cagagatggc agcgtggagg caacgcgtcc


 181
aactcctgca ctgtgctttc cttgctcgga gcccgctgtg ccttcatggg ctcgctggcc


 241
catggccatg ttgccgactt cctggtggcc gacttcaggc ggaggggtgt ggatgtgtct


 301
caagtggcct ggcagagcca gggagatacc ccttgctcct gctgcatcgt caacaactcc


 361
aatggctccc gtaccattat tctctacgac acgaacctgc cagatgtgtc tgctaaggac


 421
tttgagaagg tcgatctgac ccggttcaag tggatccaca ttgagggccg gaatgcatcg


 481
gaacaggtaa agatgctaca gcggatagaa cagtacaatg ccacgcagcc tctgcagcag


 541
aaggtccggg tgtccgtgga gatagagaag ccccgagagg aactcttcca gctgttcggc


 601
tatggagagg tggtgtttgt cagcaaagat gtggccaagc acctggggtt ccggtcagca


 661
ggggaggccc tgaagggctt gtacagtcgt gtgaagaaag gggctacgct catctgtgcc


 721
tgggctgagg agggagccga tgccctgggc cccgacggcc agctgctcca ctcagatgcc


 781
ttcccaccac cccgagtact agacactctc ggggctggag acaccttcaa tgcctctgtc


 841
atcttcagcc tctccaaggg aaacagcatg caggaggccc tgagattcgg gtgccaggtg


 901
gctggcaaga agtgtggctt gcaggggttt gatggcattg tgtgagagat gagcggtggg


 961
aggtagcagc tcgacacctc agaggctggc accactgcct gccattgcct tcttcatttc


1021
atccagcctg gcgtctggct gcccagttcc ctgggccagt gtaggctgtg gaacgggtct


1081
ttctgtctct tctctgcaga cacctggagc aaataaatct tcccctgagc c






INJURYMARKER4
INJURYMARKER4 is a 1994 nucleotide sequence encoding mitochondrial HMG-CoA Synthase [M33648]:










   1
atctctccca ggggctgtgg actgctggct ttctgttgat accttagaga tgcagcggct
(SEQ ID NO:43)



  61
tttggctcca gcaaggcggg tcctgcaagt gaagagagtc atgcaggaat cttcgctctc


 121
acccgctcac ctgctccccg acgcccagca gaggttttct acaatccctc ctgctcccct


 181
ggccaaaact gatacatggc caaaagatgt gggcatcctt gccctggagg tatactttcc


 241
agcccaatat gtggaccaaa ctgacctgga gaagttcaac aatgtggaag cagggaagta


 301
cacagtgggc ttgggccaga cccgtatggg cttctgttcg gtccaggagg acatcaactc


 361
cttgtgcctc acagtggtgc agaggctgat ggaacgcaca aagctgccat gggatgccgt


 421
aggccgcctg gaagtgggca cggaaaccat cattgacaag tccaaggctg tcaagacagt


 481
gctcatggag ctcttccagg attcaggcaa cactgacatc gagggcatag ataccaccaa


 541
cgcctgctat ggtggcactg cctccctctt caacgctgcc aactggatgg agtccagcta


 601
ctgggatggt cgctatgccc tggtggtctg tggtgatatc gcagtctacc caagtggtaa


 661
cccccgcccc acaggtggtg ccggggctgt ggcaatgctg attgggccca aggccccgct


 721
agtcctggaa caagggctga ggggaaccca catggagaac gcctatgact tctacaaacc


 781
aaacttggcc tcagagtatc cactggtgga tgggaagctg tctatccagt gctacctgcg


 841
ggccttggac cgatgctatg cagcttaccg caggaaaatc cagaatcagt ggaagcaagc


 901
tggaaacaac cagcctttca ccctcgatga cgtgcaatat atgatcttcc acacaccctt


 961
ttgcaagatg gtccagaaat ccctagctcg gctgatgttc aatgacttcc tgtcatctag


1021
cagtgacaag cagaacaact tatacaaggg tatagaggcc ttcaagggtc taaagctgga


1081
agaaacctac accaacaagg atgttgacaa ggctctgctg aaggcctccc tggacatgtt


1141
caacaagaaa accaaggcct ccctttacct ctccacaaac aatgggaaca tgtacacctc


1201
gtccctctac gggtgcctgg cctcacttct ctcccaccac tctgcccaag aattggccgg


1261
ctccaggatt ggagccttct cctacggctc aggcttagca gcaagtttct tctcatttcg


1321
agtgtccaag gacgcttccc caggttcccc tctggagaag ctggtgtcta gtgtgtcaga


1381
tctgcccaaa cgtctagact cccggagacg catgtcccct gaggaattca cagaaataat


1441
gaatcagaga gagcaatttt accacaaggt gaacttctct ccccctggtg acacaagcaa


1501
cctcttccca ggcacttggt accttgaacg agtggatgag atgcaccgca gaaaatatgc


1561
ccggcgtccc gtctaaggag accaatccat acaaccattc cccggggaaa gaatgtgagc


1621
agagccgtta cccaaacggc ttccacttaa aattccaccc acagcagtga acggtgaata


1681
gacacagcga ccccatagga tctgctccgc ggtgaagggc ctccctctgt ggatcctggg


1741
tgaccctccc tgaagcagtg agcaccacag gttctgctgt ggaccagagc ccccctgtgg


1801
agagggagaa agaaagggga gccgctgacc tgcagggata cagaccttcc ccacagcctg


1861
gcagccgccc gtttgttgca gcttattatc agactgtggg ctatcatagt tcatgctcgt


1921
ttcttaaagt ttcccgagaa tttctaaaat tttgtatcta aacttttaat atggcgatta


1981
aaaggagaga agga






INJURYMARER5
INJURYMARKER5 is a 1850 nucleotide sequence encoding cathepsin C [D90404], having the following nucleic acid sequence:










   1
gaattccggt tctagttgtt gttttctctg ccatctgctc tccgggcgcc gtcaaccatg
(SEQ ID NO:44)



  61
ggtccgtgga cccactcctt gcgcgccgcc ctgctgctgg tgcttttggg agtctgcacc


 121
gtgagctccg acactcctgc caactgcact taccctgacc tgctgggtac ctgggttttc


 181
caggtgggcc ctagacatcc ccgaagtcac attaactgct cggtaatgga accaacagaa


 241
gaaaaggtag tgatacacct gaagaagttg gatactgcct atgatgaagt gggcaattct


 301
gggtatttca ccctcattta caaccaaggc tttgagattg tgttgaatga ctacaagtgg


 361
tttgcgtttt tcaagtatga agtcaaaggc agcagagcca tcagttactg ccatgagacc


 421
atgacagggt gggtccatga tgtcctgggc cggaactggg cttgctttgt tggcaagaag


 481
atggcaaatc actctgagaa ggtttatgtg aatgtggcac accttggagg tctccaggaa


 541
aaatattctg aaaggctcta cagtcacaac cacaactttg tgaaggccat caattctgtt


 601
cagaagtctt ggactgcaac cacctatgaa gaatatgaga aactgagcat acgagatttg


 661
ataaggagaa gtggccacag cggaaggatc ctaaggccca aacctgcccc gataactgat


 721
gaaatacagc aacaaatttt aagtttgcca gaatcttggg actggagaaa cgtccgtggc


 781
atcaattttg ttagccctgt tcgaaaccaa gaatcttgtg gaagctgcta ctcatttgcc


 841
tctctgggta tgctagaagc aagaattcgt atattaacca acaattctca gaccccaatc


 901
ctgagtcctc aggaggttgt atcttgtagc ccgtatgccc aaggttgtga tggtggattc


 961
ccatacctca ttgcaggaaa gtatgcccaa gattttgggg tggtggaaga aaactgcttt


1021
ccctacacag ccacagatgc tccatgcaaa ccaaaggaaa actgcctccg ttactattct


1081
tctgagtact actatgtggg tggtttctat ggtggctgca atgaagccct gatgaagctt


1141
gagctggtca aacacggacc catggcagtt gcctttgaag tccacgatga cttcctgcac


1201
taccacagtg ggatctacca ccacactgga ctgagcgacc ctttcaaccc ctttgagctg


1261
accaatcatg ctgttctgct tgtgggctat ggaaaagatc cagtcactgg gttagactac


1321
tggattgtca agaacagctg gggctctcaa tggggtgaga gtggctactt ccggatccgc


1381
agaggaactg atgaatgtgc aattgagagt atagccatgg cagccatacc gattcctaaa


1441
ttgtaggacc tagctcccag tgtcccatac agctttttat tattcacagg gtgatttagt


1501
cacaggctgg agacttttac aaagcaatat cagaagctta ccactaggta cccttaaaga


1561
attttgccct taagtttaaa acaatccttg atttttttct tttaatatcc tccctatcaa


1621
tcaccgaact acttttcttt ttaaagtact tggttaagta atacttttct gaggattggt


1681
tagatattgt caaatatttt tgctggtcac ctaaaatgca gccagatgtt tcattgttaa


1741
aaatctatat aaaagtgcaa gctgcctttt ttaaattaca taaatcccat gaatacatgg


1801
ccaaaatagt tattttttaa agactttaaa ataaatgatt aatcgatgct






INJURYMARKER6
INJURYMARKER6 is a 993 nucleotide sequence encoding hydroxysteroid sulfotransferase [D14989]:










  1
ggcaagggct ggaatactaa aagttattca tgatgtcaga atatacttgg tttgaaggaa
(SEQ ID NO:45)



 61
taccttttcc tgccttttgg ttttccaaag aaattctgga aaatagttgt aagaagtttg


121
tggtaaaaga agacgacttg atcatattga cttaccccaa gtcaggaacg aactggctga


181
tcgagattgt ctgcttgatt cagaccaagg gagatcccaa gtggatccaa tctatgccca


241
tctgggatcg ctcaccctgg atagagactg gttcaggata tgataaatta accaaaatgg


301
aaggaccacg actcatgacc tcccatcttc ccatgcatct tttctccaag tctctcttca


361
gttccaaggc caaggtgata tatctcatca gaaatcccag agatgttctt gtttctgctt


421
attttttctg gagtaagatc gccctggaga agaaaccaga ctcgctggga acttacgttg


481
aatggttcct caaaggaaat gttgcatatg gatcatggtt tgagcacatc cgtggctggc


541
tgtctatgag agaatgggac aacttcttgg tactgtacta tgaagacatg aaaaaggata


601
caatgggatc cataaagaag atatgtgact tcctggggaa aaaattagag ccagatgagc


661
tgaatttggt cctcaagtat agttccttcc aagtcgtgaa agaaaacaac atgtccaatt


721
atagcctcat ggagaaggaa ctgattctta ctggttttac tttcatgaga aaaggcacaa


781
ctaatgactg gaagaatcac ttcacagtag cccaagctga agcctttgat aaagtgttcc


841
aggagaaaat ggccggtttc cctccaggga tgttcccatg ggaataaatt ttcaaaagtt


901
ttaaatattt tatgaacact gatgtttatg tttatgttgt tctatgatgt ctgaataact


961
gaatgtgatc attgaataaa tcctgttgtg gat






INJURYMARKER7
INJURYMARKER7 is a 5001 nucleotide sequence encoding insulin-like growth factor binding protein [L22979]:










   1
cacaaaccca gcgagcattg aacactgcac acggccatct gcccagagag ctgtgaccac
(SEQ ID NO:46)



  61
cacttccgct actatctact cagaaagtcg tgactactga gccactgctg cctgcccaga


 121
ttctcatcca ccgcctgctg cgtctggttg cgatgccgga gttcctaact gttgtttctt


 181
ggccgttcct gatcctcctg tccttccagg ttcgcgtagt cgctggagcc ccccagccat


 241
ggcactgtgc tccctgcact gctgagaggc tggagctctg tccacccgtg cctgcttcgt


 301
gccccgagat ttctcggcct gcgggctgtg gctgctgccc gacatgtgcc ttgccactgg


 361
gtgctgcctg tggtgtggcc actgcggcct gcgctcaggg actcagctgc cgtgcgctgc


 421
caggggagcc tcgacctctg catgccctca cccgtggcca gggagcctgt gtactagaac


 481
ctgccgcacc cgccacgagc agcttgtccg gttctcagca tgaaggtact acagccctct


 541
ctgcctcttg atctcttggc taggacacac gtgctttcta ggcacgtcag aggcctatcc


 601
ggaacctata gcagatagga caaaggctct ccatgcccac tttgagcttt cagcctcaaa


 661
taaggccctc agttaggtcg tggcggcttg ggaaacacca gaggtgtcaa tccagtagca


 721
gagtggagaa gttgggaaga atgttccaag ctcccagtgc agagtggaga gttgggaaga


 781
atgttcacag actaggtagt actgatcctg cttggtcttt cagtggggag ggagctatgg


 841
ggctgccagg tgggtggggt gctggcccaa acacctcttt ctgtgggtcc tgaccttggc


 901
agttccaatg gctaaaaggt ccaggaaggt ttaggatggg agccctcctg ctgcccccag


 961
gaggtttgca atgtcctttg tagcatatat cctgccacac agtatgtgct tcccagatgt


1021
ttacagaaca taatgtgaaa atttaggccc aaaccttcac ttccattcat tgctatagac


1081
aaacagtgtt tgaagtgtat gttgcctgct aggagtctga caatcaggcg ctttcctgaa


1141
tttaagcact ggtttgtttg taataggaag cttgggaaat gcctcttcct ctgctccagc


1201
gggtatctcc cctgtctggg ctgcatgcac ttcctgtgtg ggtaagggac ctcatggttc


1261
catattctga cgggaagccg gactgcaggc atctgatcct tttgactaaa tggaagaact


1321
atcccaacgg tccttagaaa cgggcttccc caggagcgat gtctgataat gtcctcctct


1381
gtgaggggct gcctaagagg tgtcggtgtt caagaaagca gggctcccag aaaagaagag


1441
gatggtggtg tgaggtgggg aaggctacac tctacacctt gcttctcaac tatcccctta


1501
ctggggtctt acgagattct ttttgtggtg tggagaggag agctgagtgg tcaagtctca


1561
ccactaacgg gttcaagcct tggcctcagt ccttggcttc ttcaggatta catcctagac


1621
ccaactctct ctgccatggg gactcccttg cctaacccca aaacatacca tttccccaga


1681
aaggaattag tattgctaat tggtgataat tgttcccaaa tagcccactg gtgaaaacaa


1741
agcctgattt cacctgactg ttacagattg gtcttaaggc ggtagacgtg agtgacatag


1801
gagtgacacc tcagggctca tcgtctgtgt ctgtggggtt cgttttcaga ggcaaaggct


1861
gctgtggcct ctgaggatga gcttgccgag agcccagaga tgacagagga acagctgctg


1921
gatagcttcc acctcatggc cccatcccgt gaggaccagc ccatcctgtg gaatgccatt


1981
agcacctaca gcagcatgcg ggcccgggag atcactgacc tcaagaaatg gaaggtgaga


2041
ccctgcactc agaccttcag gtttagctat ctacgtgaag aggtttgtct agacgatttc


2101
ttaaagggca ctgagcatgg ggctgagaac ggggatataa ctacccccat ccctgatgta


2161
tttctgcctc cttaaaaata tggcaagtat ctcagagcat aaggtaggcc atttttcagt


2221
ctaggtttct ctgtcaccga gtacgcacgt tcagtgattg ttagccacca accagctcca


2281
cggttttgcc agcctttagc tatgcacttt agctatgcag taaacttctc tagctttact


2341
ggctgttttt caacttgacc acttggggga gacagagaac caaaggtgga gagaaagtac


2401
ggcagaggca ttgaagaagt acacctaagg aaatgaaagg ataaacattg ttaggggcac


2461
tttagaattt catatggaaa ttgtccaaat cagtgccttg ttccgtaatc aatttgacat


2521
acaccaaatg caaggatggc tgtttgaaaa atctaggcat ttatgatgct aaattccaca


2581
cacagagact gagcctgtct tttttattag agttcaggtg ctcaagttat tcagagatag


2641
ccagggtcag gaagcattta taccattggc caggctctta ccacaatgtc gttaaggggg


2701
tctccagaaa atgccactga gggaggatga gagtggtgtc cctgtccttt atctacatag


2761
cccaagccag agaccaacct gtcctgctca cagatgggga aacatctcag ccgttgtcta


2821
aattgataat ttttgtctct tgtactcatg ctaatataaa attatccttt taggagccct


2881
gccaacggga actctataaa gtgttagaga gattagctgc cgctcaacag aaagcaggag


2941
atgagatcta caaattttat ctgccaaact gcaacaagaa tggattttat cacagcaaac


3001
aggtaggtgg ctttgctcat ccagatcctt gtaaaacttc atgatttttt tttttaaagt


3061
caaatgattc acaggcccaa tacacatcat gggtagcttt cttaggtgag atccagccct


3121
gcagtagttg ggagaagcta gtcctgagaa agagatagtg tgatggatga ggaacacttc


3181
agccagaagg gaggactaag cattagtgtg atgagtgagg agcacttcag ttaacaggga


3241
ggactaagca ttagtgtgat gagtgaggac cacttcaagc cagagggagg actaacattg


3301
gcagtatgat gagtgaggag cacttcagcc agtagggagg actaaccatt agtctcatca


3361
ctaggagcac ctcagccaag tagggaggac taaccattag tctcacactc acccaacatc


3421
ttcagtcagg actaagcatt agtgtgatga gtgaggagca cttcagtcag tagggaggac


3481
tacattagtg tgatgagtga ggagcacttc agccagtagg gaggactaac cgttcactca


3541
gattagcaga gatggatgtt ccatatactg atgtccaggt ttcagttcct cacaactaga


3601
ggaaagggac acagtcagtg taggagacag atgtatcgcg ttctctcttc ccacaaataa


3661
aaacaaactc tgtagtaaga cacaccaatt gtgctttgcc tagcaataaa tgagattgaa


3721
gaagtccagg cttaatttcg acgcaacttt agaactcagg gaagtgcaag ttctggaatt


3781
tcattgagga aaaacttgag gtctaggtct agccgtgtgg tagagatggt gagacctatc


3841
gttgagctcc tttggcagag ggccatggag caggtaaccg tcaaaacaat ataccactga


3901
gtaaacagat gagattgtta tcaggtgtgc cataaagcca acctctccgt tttgtgatga


3961
caaccagaag ggcattggtc tgccgagcct tagccagcag gtagctgtgc agtgcttggc


4021
ctcactgagg gacagggtgg ccagagctct tacctcctgc tgctcttgac ctcggtcctg


4081
tctttgcagt gcgagacatc tctggatgga gaagctgggc tctgctggtg tgtctaccca


4141
tggagtggga agaagatccc tggatctctg gagaccagag gggaccccaa ctgccaccag


4201
tattttaatg tgcaaaactg aaagttgttt cctccctcct tcttcacaca aaatatttaa


4261
gtatatagtg tatttatact ccggagcaca ccattttata tatgtgtata tgtatatatc


4321
caggaactag tttttatact ccacatgctg cttgatgtac aagtgggttt gtatttattc


4381
actctaagtt tatttttttc taccctgtcc ttgtgctgta ttaatttata taactgaagc


4441
ttttctcatc tccatacatg taaatactac catctcagct cttccagagt tctgctttga


4501
aagggcagcg cggtacgtgc ctagaacgag cacaagtcag tctgaggtag gggcctttca


4561
gtgggttcag ggaggaaggt tagccctggc tcggggagac ttcctcatcg aatcccacag


4621
gtctgtgtct gatgcctatt ggctgggaag gttccgatgt tggttgtgta atcaaagcta


4681
aagctggaaa gctgcgtccc atgcactgtt aaacacacgt ctggaatccc acattctacc


4741
tggaaacact gctgtctctg tggaattcca gctctgtgct cattccctca gtccgttcgg


4801
ctttcccgct cgcctgattc ctgggtctgt gctttgggga tagatgttgc aatacagggt


4861
gcttgtttgt ttacagaaca ccctggacaa acactctgtg actttatggt cccattttca


4921
agcagcatca ggcctctgtc tgggccagac tacagagccc ctcctccttg gtccatctcc


4981
ctttcttccc agggccctca g






INJURYMARKER8
INJURYMARKER8 is 579 bp rat expressed sequence tag (EST) [AA851963]. The acid was initially identified in a cloned fragment having the following sequence:










  1
TGTACATAATTTATTAAAAATGTCTCTGACACAAATAATGACTCCACTGCATACATAGTTGGTGTTCAAAAATTTCCCCA
(SEQ ID NO:13)



 81
ATGTTTGTTCTGGACACAATTGTTATTAGCCAACTCGGTGAATTCAAGACATTGTTCCACACAATGAACAATCGCACACA


161
TGAGAACTGCACCTAGAATGTCCATCCTAGAATCTCCATCCATCCAGTCAAAGTGCTGAGCTCACTGACTGAAGGAAACA


241
TGACCTGTGTTCTAGA






The cloned sequence was assembled into a contig resulting in the following consensus sequence:










  1
TGTACATAATTTATTAAAAATGTCTCTGACACAAATAATGACTCCACTGCATACATAGTTGGTGTTCAAAAATTTCCCCA
(SEQ ID NO:14)



 81
ATGTTTGTTCTGGACACAATTGTTATAAGCCAACTCGGTGAATTCAAGACATTGTTCCACACAATGAACAATCGCACACA


161
TGAGAACTGCACCTACAATGTCCATCCTAGAATCTCCATCCATCCAGTCAAAGTGCTGAGCTCACTGACTGAAGGAAACA


241
TGACCTGTGTTCTAGAACGTAGCTGGCTATGAAGTTTACTCATGTGTAAATTCCTTAAAAAGATTAAATTGTTTGGCCCA


321
TTTCTATATTTCATAAAATAACTATAATTACAAACTTTCTAAAAATAATTTTACAACCATGTAATTATGACTAACCATAT


401
CATCTAAAAAGTAAGTGAAGTCATTGTCCTAGAGATTGTCTGAGATTATTCTGCTGAGAAGCTTACTTCAAACTCTTATC


481
ACTACTTCCTACTTCCAGTGTCCTTGAATTAAGAACAGAAATTGTAACTATGCTATTCTACATCAGATTGACACAACCTA


561
CTTCTAAGTACACTATTGC






Blast-N Results:

>gb:GENBANK-ID:AA851963|acc:AA851963 EST194732 Normalized rat spleen, Bento Soares Rattus sp. cDNA clone RSPA086 3′ end, mRNA sequence—Rattus sp., 538 bp (RNA).

Top Previous Match Next MatCh

    
    
        Length=538

Plus Strand HSPs:

    
    










Score = 2681 (402.3 bits), Except = 8.1e-115, p = 8.1e-115



Identities = 537 538 (99%), Positives = 537/538 (99%), Strand = Plus/Plus











Query:
 42
CTCCACTGCATACATAGTTGGTGTTCAAAAATTTCCCCAATGTTTGTTCTGGACACAATT
101
(SEQ ID NO:47)




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
  1
CTCCACTGCATACATAGTTGGTGTTCAAAAATTTCCCCAATGTTTGTTCTGGACACAATT
60
(SEQ ID NO:48)


Query:
102
GTTATAAGCCAACTCGGTGAATTCAAGACATTGTTCCACACAATGAACAATCGCACACAT
161




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
 61
GTTATTAGCCAACTCGGTGAATTCAAGACATTGTTCCACACAATGAACAATCGCACACAT
120


Query:
162
GAGAACTGCACCTAGAATGTCCATCCTAGAATCTCCATCCATCCAGTCAAAGTGCTGAGC
221




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
121
GAGAACTGCACCTAGAATGTCCATCCTAGAATGTCCATCCATCCAGTCAAAGTGCTGAGC
180


Query:
222
TCACTGACTGAAGGAAACATGACCTGTGTTCTAGAACGTAGCTGGCTATGAAGTTTACTC
281




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
181
TCACTGACTGAAGGAAACATGACCTGTGTTCTAGAACGTAGCTGGCTATGAAGTTTACTC
240


Query:
282
ATGTGTAAATTCCTTAAAAAGATTAAATTGTTTGGCCCATTTCTATATTTCATAAAATAA
341




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
241
ATGTGTAAATTCCTTAAAAAGATTAAATTGTTTGGCCCATTTCTATATTTCATAAAATAA
300


Query:
342
CTATAATTACAAACTTTCTAAAAATAATTTTACAACCATGTAATTATGACTAACCATATC
401




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
301
CTATAATTACAAACTTTCTAAAAATAATTTTACAACCATGTAATTATGACTAACCATATC
360


Query:
402
ATCTAAAAAGTAAGTGAAGTCATTGTCCTAGAGATTGTCTGAGATTATTCTGCTGAGAAG
461




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
361
ATCTAAAAAGTAAGTGAAGTCATTGTCCTAGAGATTGTCTGAGATTATTCTGCTGAGAAG
420


Query:
462
CTTACTTCAAACTCTTATCACTACTTCCTACTTCCAGTGTCCTTGAATTAAGAACAGAAA
521




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
421
CTTACTTCAAACTCTTATCACTACTTCCTACTTCCAGTGTCCTTGAATTAAGAACAGAAA
480


Query:
522
TTGTAACTATGCTATTCTACATCAGATTGACACAACCTACTTCTAAGTACACTATTGC
579




||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


Sbjct:
481
TTGTAACTATGCTATTCTACATCAGATTGACACAACCTACTTCTAAGTACACTATTGC
538






INJURYMARIKER9
INJURYMARKER9 is a 2495 nucleotide catalese-encoding sequence[M11670], having the following nucleic acid sequence:










   1
attgcctacc ccgggtggag accgtgctcg tccggccctc ttgcctcacg ttctgcagct
(SEQ ID NO:49)



  61
ctgcagctcc gcaatcctac accatggcgg acagccggga cccagccagc gaccagatga


 121
agcagtggaa ggagcagcgg gcccctcaga aacccgatgt cctgaccacc ggaggcggga


 181
acccaatagg agataaactt aatatcatga ctgcggggcc ccgagggccc ctcctcgttc


 241
aagatgtggt tttcaccgac gagatggcac actttgacag agagcggatt cctgagagag


 301
tggtacatgc aaagggagca ggtgcttttg gatactttga ggtcacccac gatattacca


 361
gatactccaa ggcaaaggtg tttgagcata ttgggaagag gactcctatt gccgtccgat


 421
tctccacagt cgctggagag tcaggctcag ctgacacagt tcgtgaccct cgtgggtttg


 481
cagtgaaatt ctacactgaa gatggtaact gggacctcgt gggaaacaac acccctattt


 541
tcttcatcag ggatgccatg ttgtttccat cctttatcca tagccagaag agaaacccac


 601
aaactcacct gaaggaccct gacatggtct gggacttctg gagtctttgt ccagagtctc


 661
tccatcaggt tactttcttg ttcagcgacc gagggattcc agatggacat cggcacatga


 721
atggctatgg ctcacacacc ttcaagctgg ttaatgcgaa tggagaggca gtgtactgca


 781
agttccatta caagactgac cagggcatca aaaacttgcc tgttgaagag gcaggaagac


 841
ttgcacagga agacccggat tatggcctcc gagatctttt caatgccatc gccagtggca


 901
attacccatc ctggactttt tacatccagg tcatgacttt caaggaggca gaaaccttcc


 961
catttaatcc atttgacctg accaaggttt ggcctcacaa ggactaccct cttataccag


1021
ttggcaaact ggtcttaaac agaaatcctg ctaattattt tgctgaagtt gaacagatgg


1081
cttttgaccc aagcaacatg ccccctggca ttgagcccag cccggacaag atgctccagg


1141
gccgcctttt tgcttaccca gacactcacc gccaccgcct gggaccaaac tatctgcaga


1201
tacctgtgaa ctgtccctac cgtgctcgcg tggccaacta ccagcgcgat ggccccatgt


1261
gcatgcatga caaccagggt ggtgctccca actactaccc caacagcttc agcgcaccag


1321
agcagcaggg ctcggccctg gagcaccata gccagtgctc tgcagatgtg aagcgcttca


1381
acagtgctaa tgaagacaac gtcactcagg tgcggacatt ctatacgaag gtgttgaatg


1441
aggaggagag gaaacgcctg tctgagaaca ttgccaacca cctgaaagat gctcagcttt


1501
tcattcagag gaaagcggtc aagaatttca ctgacgtcca ccctgactac ggggcccgag


1561
tccaggctct tctggaccag tacaactccc agaagcctaa gaatgcaatt cacacctacg


1621
tacaggccgg ctctcacata gctgccaagg gaaaagctaa cctgtaaagc acgggtgctc


1681
agcctcctca gcctgcactg aggagatccc tcatgaagca gggcacaagc ctcaccagta


1741
atcatcgctg gatggagtct cccctgctga agcgcagact cacgctgacg tctttaaaac


1801
gataatccaa gcttctagag tgaatgatag ccatgctttt gatgacattt cccgaggggg


1861
aaattaaaga ttagggctta gcaatcactt aacagaaaca tggatctgct taggacttct


1921
gtttggatta ttcatttaaa atgattacaa gaaaggtttt ctagccagaa acatgatttg


1981
attagatatg atatatgata aaatcttggt gattttacta tagtcttatg ttacctcaca


2041
gaatggtata tatacaacac acacacacac acacacacac acacaccaaa acacacatac


2101
actatacaca acacacacac acacacataa aacacacata cacaacacac acatacacta


2161
cacacacaga acacacaaca caaacataca cacataggca cacacacaca cacacacaca


2221
cacacacaca cacacacaca cacacatgaa tgaagggatt ataaagatgg cccacccaga


2281
attttttttt atttttctaa gatccttata agaaaaacca tacttggatc atgtcttcca


2341
aaaataactt tagcactgtt gaaacttaat gtttattcct gtgtagttga ttggattcct


2401
tttccccttg aaattatgtt tatgctgata cacagtgatt tcacataggg tgatttgtat


2461
ttgcttacat ttttacaata aaatgatctt catgg






INJURYMARKER10
INJURYMARKER10 is a 1884 nucleotide betaine homocysteine methyl transferase-encoding sequence [AF038870]:










   1
caagcctttg ctggagaccg ctcctgtcca gtccgcagct ggcttcagcg ccactcagga
(SEQ ID NO:50)



  61
caccggaaag atggcaccga ttgccggcaa gaaggccaag aggggaatct tagaacgctt


 121
aaatgctggc gaagtcgtga tcggagatgg gggatttgtc tttgcactgg aaaagagggg


 181
ctacgtaaag gctggaccct ggaccccaga ggctgcggtg gagcaccccg aggcagttcg


 241
gcagcttcat cgggagttcc tcagagctgg atcgaacgtc atgcagacct tcactttcta


 301
tgcaagtgag gacaagctgg aaaaccgagg gaactacgtg gcagagaaga tatctgggca


 361
gaaggtcaat gaagctgctt gtgacattgc acggcaagtt gctgacgaag gggatgcatt


 421
ggttgcagga ggtgtgagtc agacaccttc ctacctcagc tgcaagagtg agacggaagt


 481
taaaaagata tttcaccaac agcttgaggt cttcatgaag aagaatgtgg acttcctcat


 541
tgcagagtat tttgaacatg ttgaagaagc cgtgtgggca gtcgaggcct taaaaacatc


 601
cgggaagctt atagcggcta ccatgtgcat cggacctgaa ggagatctac atggcgtgtc


 661
tcctggagag tgcgcagtgc gtttggtaaa agcaggtgcc gccattgtcg gtgtgaactg


 721
ccacttcgac cccagcacca gcttgcagac aataaagctc atgaaggagg gtctggaagc


 781
agctcggctg aaggcttact tgatgagcca cgccctggcc taccacaccc ctgactgtgg


 841
caaacaggga tttattgatc tcccagaatt cccctttgga ttggaaccca gagttgccac


 901
cagatgggat attcaaaaat acgccagaga ggcctacaac ctgggggtca ggtacattgg


 961
cggctgctgc ggatttgagc cctaccacat cagggccatt gcagaggagc tcgccccaga


1021
aaggggattt ttaccaccag cttcagaaaa acatggcagc tggggaagtg gtttggacat


1081
gcacaccaaa ccctggatca gggcaagggc caggaaagaa tactggcaga atcttcgaat


1141
agcttcgggc agaccgtaca atccttcgat gtccaagccg gatgcttggg gagtgacgaa


1201
aggggcagca gagctgatgc agcagaagga agccaccact gagcagcagc tgagagcgct


1261
cttcgaaaaa caaaaattca aatccgcaca gtagccacag gccagcggtt cggggcgaat


1321
tcctccaggt ccgggccaca gtgtgcaccc ggaaggagaa ggcatctcta aaccagcgtt


1381
tgtgttgatg ccggcttaca cctgtgattg gtgctagtta gacaaaatgg agtcacagat


1441
agcatttcac agttacaaaa ctacgcttta gaattttacc tagaaggaag aaaggagaag


1501
tccacagtaa atcctgaaca catttcctac gtgcctgtcg cattacaggc gcacaggagt


1561
cactgcagcg aagagaaagt cacccgacgt caatctcatt tcagataggg ggataggaca


1621
ccacctccac gagtgacata gaaccattca gggaccgtat cataagtgac acagcaacca


1681
tctatatcta agatgcttcc caagtggatt ccaagatctt ttgagcagga cccttaggca


1741
gaaacaacac acaccagccc tgtaaaactt aacagataac tgatccattc tgtaattctg


1801
taatctctgt tctgactgct tccattccat ttcattaata aaaacatgcc ggttgaaaac


1861
cttcaaaaaa aaaaaaaaaa aaa






Principle components analysis was used to generate three eigenvectors used to transform the original expression level data matrix, as shown in Table 6 below. Eigenvector 1 values represent NSAIDs associated with hepatoxicity involving hepatocellular damage, Eigenvector 2 values represent NSAIDs associated with hepatoxicity involving cholestasis, and Eigenvector 3 values represent NSAIDs associated with hepatoxicity involving elevated transaminase level.







TABLE 6







Transform Eigenvectors for Hepatoxicity by Injury Type










Gene
Eigenvector 1
Eigenvector 2
Eigenvector 3













INJURYMARKER 1
58.7
0.325
−15.2


INJURYMARKER 2
205
−3.23
3.01


INJURYMARKER 3
−16.9
−6.52
−2.09


INJURYMARKER 4
−10.3
0.351
−1.45


INJURYMARKER 5
−7.59
−0.152
−0.310


INJURYMARKER 6
11.4
−2.69
2.49


INJURYMARKER 7
−16.0
−1.57
8.71


INJURYMARKER 8
−11.6
1.13
5.36


INJURYMARKER 9
−11.0
−0.351
0.078


INJURYMARKER 10
7.55
0.618
4.65


% of variation
99.0
0.7
0.3


explained









These eigenvectors may be used to transform the expression levels of INJURYMARKERS 1-10 (“INJURYMARKERS”) in response to a given drug, in order to predict that drug's hepatotoxicity injury type. For example, expression levels of INJURYMARKERS correlating with Eigenvector 1 indicates that the test drug has a risk of hepatotoxicity involving hepatocellular damage. Alternatively, a drug's INJURYMARKERS expression profile can be generated simultaneously with the above-described training set (or an equivalent set) run in parallel with the test drug, and expression levels associated with the test drug directly compared to those of the training set.
General Methods
The RISKMARKER (i. e. RISKMARKERS 1-8) and INJURYMARKER (i e. INJURYMARKERS 1-10) nucleic acids and encoded polypeptides can be identified using the information provided above. In some embodiments, the RISKMARKER or INJURYMARKER nucleic acids and polypeptide correspond to nucleic acids or polypeptides which include the various sequences (referenced by SEQ ID NOs) disclosed for each RISKMARKER or INJURYMARKER 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 RISKMARKER 1-8 or INJURYMARKER 1-10. 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 RISKMARKER or INJURYMARKER 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 RISKMARKER or INJURYMARKER 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 RISKMARKER or INJURYMARKER sequences, or within the sequences disclosed herein, can be used to construct probes for detecting RISKMARKER or INJURYMARKER 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 RISKMARKER or INJURYMARKER sequences in, e.g., amplification-based detection methods such as reverse-transcription based polymerase 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 RISKMARKER or INJURYMARKER sequences in the test cell population, e.g. rat hepatocytes, 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 example, expression can be compared using GENECALLING® methods as described in U.S. Pat. No. 5,871,697 and in Shimkets et al., Nat. Biotechnol. 17:798-803.
In various embodiments, the expression of one or more sequences which are markers of hepatoxicity risk, i.e. RISKMARKERS 1-8, is compared. In other embodiments, the expression of one or more sequences which are markers of hepatoxicity injury type, ie. INJURYMARKERS, is compared. In further embodiments, expression of one or more RISKMARKERS and INJURYMARKERS may be compared to predict both hepatoxicity risk and type of hepatoxicity injury.
In various embodiments, the expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or all of the sequences represented by RISKMARKER 1-8 and INJURYMARKER 1-10 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 thiazolidinedione such as troglitazone. 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 RISKMARKER or INJURYMARKER sequence in a test cell population is considered comparable in expression level to the expression level of the RISKMARKER or INJURYMARKER sequence if its expression level varies within a factor of 2.0, 1.5, or 1.0 fold to the level of the RISKMARKER or INJURYMARKER transcript in the reference cell population. In various embodiments, a RISKMARKER or INJURYMARKER sequence in a test cell population can be considered altered in levels of expression if its expression level 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 RISKMARKER or INJURYMARKER sequence in the reference cell population.
Alternatively, the absolute expression level matrix of the 8 RISKMARKER and/or 10 INJURYMARKER fragments in a test cell can be transformed using the principal component eigenvectors described above, or similar eigenvalues generated from parallel dosed members of the training set as internal controls. The expression eigenvalues for the test cell can then be compared to the training set eigenvalues described herein, or a parallel-run training set, if any.
The RISKMARKER expression level combination is considered similar to Low Risk idiosyncratic NSAIDS (several of which have been withdrawn), if the test drug's expression profile is within the 95% confidence interval (CI) of the centroid of that risk class. See Table 4. The test drug is considered Very Low Risk idiosyncratic if the transformed expression profile falls within the 95% CI of the centroid of that class. The test drug is considered Overdose Risk if the expression profile falls within the 95% CI of the centroid of that class. If the compound fails to associate with any of these compounds it will be categorized as unclassifiable.
Similarly, the INJURYMARKER expression level combination is considered indicative of hepatocellular damage induced by idiosyncratic NSAIDS, if the test drug's expression profile is within the 95% confidence interval (CI) of the centroid of that injury type. See Table 6. The test drug is considered to induce idiosyncratic cholestasis if the transformed expression profile falls within the 95% CI of the centroid of that injury type. The test drug is considered to induce elevated transaminase level if the expression profile falls within the 95% CI of the centroid of that class. If the compound fails to associate with any of these compounds it will be categorized as unclassifiable.
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.
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 of RISKMARKER 1-8 or INJURYMARKER 1-10. The sequences need not be identical to sequences including RISKMARKER or INJURYMARKER nucleic acid sequences, 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 RISKMARKER or INJURYMARKER nucleic acids described herein.
Expression of the nucleic acid sequences in the test cell population is then compared to is 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, e.g. a NSAID such as ketoprofen.
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.
In some embodiments of the method of the invention, the test agent is an idiosyncratic hepatotoxic agent, e.g. a NSAID, and the reference agent is also a NSAID. As described above, RISKMARKER (e.g. RISKMARKERS 1-8) expression level patterns can be used to predict the level of hepatoxicity risk (i.e. low, very low, or overdose) associated with a given test agent, e.g. a NSAID. In one embodiment, the reference NSAID (ie. used with the reference cell population) is a NSAID classified as having a low risk of hepatoxicity. The test agent is then identified as having a low risk of hepatoxicity if no qualitative difference in expression levels is identified in comparison to expression levels in the reference population exposed to a low risk NSAID. In certain embodiments, the low risk NSAID is Benoxaprofen, Bromfenac, Diclofenac, Phenylbutazone, or Sulindac. In another embodiment, the reference NSAID is a NSAID classified as having a very low risk of hepatoxicity. The test agent is then identified as having a very low risk of hepatoxicity if no qualitative difference in expression levels is identified in comparison to expression levels in the reference population exposed to a very low risk NSAID. In certain embodiments, the very low risk NSAID is Etodolac, Fenoprofen, Flurbiprofen, Ibuprofen, Indomethacin, Ketoprofen, Meclofenamate, Mefenamic Acid, Nabumetone, Naproxen, Oxaprozin, Piroxicam, Suprofen, Tenoxicam, Tolmentin, or Zomepirac. In still another embodiment, the reference NSAID is a NSAID classified as having an overdose risk of hepatoxicity. The test agent is then identified as having an overdose risk of hepatoxicity if no qualitative difference in expression levels is identified in comparison to expression levels in the reference population exposed to an overdose risk NSAID. In certain embodiments, the overdose risk NSAID is Acetaminophen, Acetylsalicylic acid, or Phenacetin. In some embodiments, the difference in expression levels is determined by comparing expression transformation eigenvectors (for risk class) for the test cell and reference cell populations, as described above.
As also described above, INJURYMARKER (e.g. INJURYMARKERS 1-10) expression level patterns can be used to predict the type of hepatoxicity injury (i.e. hepatocellular damage, cholestasis, or elevated transaminase level) associated with a given test agent, e.g. a NSAID. In some embodiments, the reference NSAID is a NSAID classified as inducing hepatocellular damage. The test agent is then identified as likely to induce hepatocellular damage if no qualitative difference in expression levels is identified in comparison to expression levels in the reference population exposed to a NSAID:) which induces hepatocellular damage. In certain embodiments, the hepatocellular damage inducing NSAID is Acetaminophen, Flurbiprofen, or Ketoprofen. In another embodiment, the reference NSAID is a NSAID classified as inducing cholestasis. The test agent is then identified as likely to induce cholestasis if no qualitative difference in expression levels is identified in comparison to expression levels in the reference population exposed to a NSAID which induces cholestasis. In certain embodiments, the cholestatis-inducing NSAID is Benoxaprofen, Nabumetone, or Sulindac. In yet another embodiment, the reference NSAID is a NSAID classified as inducing elevated transaminase level. The test agent is then identified as likely to induce elevated transaminase level if no qualitative difference in expression levels is identified as compared to expression levels in the reference population exposed to a NSAID which induces elevated transaminase levels. In certain embodiments, the elevated transaminase level inducing NSAID is Zomepirac, Mefenamic acid, or Tenoxicam. In some embodiments, the difference in expression levels is determined by comparing expression transformation eigenvectors for said test cell and reference cell populations, as described above.
Assessing Toxicity of a Toxic Agent in a Subject
The differentially expressed RISKMARKER or INJURYMARKER sequences identified herein also allow for the hepatotoxicity of a hepatotoxic agent to be determined or monitored. In this method, a test cell population from a subject is exposed to a test agent, ie. 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 RISKMARKER or INJURYMARKER sequences 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 RISKMARKER or INJURYMARKER sequences in the test cell population and the reference cell population indicates that the treatment is non-hepatotoxic. However, a difference in expression between RISKMARKER or INJURYMARKER 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 leads to liver damage.
As described in detail above, RISKMARKER expression patterns can be used to predict the level of hepatotoxicity risk (e.g. low risk, very low risk, overdose risk) associated with a test agent or drug, by comparison to RISKMARKER expression levels for reference drugs, e.g. NSAIDs, with a given classification of risk (e.g. very low risk). Similarly, INJURYMARKER expression patterns can be used to predict the type of hepatotoxicity damage (e.g. hepatocellular damage, cholestasis, elevated transaminase level) associated with a test agent or drug, by comparison to INJURYMARKER expression levels for reference drugs, e.g. NSAIDs, which induce a given type of hepatotoxic damage (e.g. cholestasis).
Riskmarker Nucleic Acids
Also provided in the invention are novel nucleic acid comprising a nucleic acid sequence selected from the group consisting of RISKMARKER 1, and RISKMARKERS 6-8, or their complements, as well as vectors and cells including these nucleic acids.
Thus, one aspect of the invention pertains to isolated RISKMARKER nucleic acid molecules that encode RISKMARKER proteins or biologically active portions thereof. Also included are nucleic acid fragments sufficient for use as hybridization probes to identify RISKMARKER-encoding nucleic acids (e.g., RISKMARKER mRNA) and fragments for use as polymerase chain reaction (PCR) primers for the amplification or mutation of RISKMARKER 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.
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 (ie., 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 RISKMARKER nucleic acid molecule can contain less than about 50 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 RISKMARKER 1, or RISKMARKER 6-8, 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, RISKMARKER or INJURYMARKER nucleic acid sequences can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., eds., Molecular Cloning: A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 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, oligonucleotide corresponding to RISKMARKER 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 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 RISKMARKER 1, or RISKMARKER 6-8. 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 RISKMARKER 1, or RISKMARKER 6-8 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 RISKMARKER 1, or RISKMARKER 6-8, e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically active portion of RISKMARKER. 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 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, N.Y., 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, Wis.) 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 RISKMARKER 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 RISKMARKER polypeptide of species other than humans, including, but not limited to, mammals, and thus 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 RISKMARKER protein. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in a RISKMARKER polypeptide, as well as a polypeptide having a RISKMARKER activity. A homologous amino acid sequence does not encode the amino acid sequence of a human RISKMARKER polypeptide.
The nucleotide sequence determined from the cloning of human RISKMARKER genes allows for the generation of probes and primers designed for use in identifying and/or cloning RISKMARKER homologues in other cell types, e.g., from other tissues, as well as RISKMARKER 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 RISKMARKER sequence, or an anti-sense strand nucleotide sequence of a nucleic acid comprising a RISKMARKER sequence, or of a naturally occurring mutant of these sequences.
Probes based on human RISKMARKER 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 RISKMARKER protein, such as by measuring a level of a RISKMARKER-encoding nucleic acid in a sample of cells from a subject e.g., detecting RISKMARKER mRNA levels or determining whether a genomic RISKMARKER gene has been mutated or deleted.
“A polypeptide having a biologically active portion of RISKMARKER” 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 RISKMARKER” can be prepared by isolating a portion of RISKMARKER 1, or RISKMARKER 6-8, that encodes a polypeptide having a RISKMARKER biological activity, expressing the encoded portion of RISKMARKER protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of RISKMARKER. For example, a nucleic acid fragment encoding a biologically active portion of a RISKMARKER polypeptide can optionally include an ATP-binding domain. In another embodiment, a nucleic acid fragment encoding a biologically active portion of RISKMARKER includes one or more regions.
Riskmarker and Injurymarker Variants
The invention further encompasses nucleic acid molecules that differ from the disclosed or referenced RISKMARKER or INJURYMARKER nucleotide sequences due to degeneracy of the genetic code. These nucleic acids thus encode the same RISKMARKER or INJURYMARKER protein as that encoded by nucleotide sequence comprising a RISKMARKER or INJURYMARKER nucleic acid as shown in, e.g., RISKMARKER 1-8 INJURYMARKER 1-10.
In addition to the rat RISKMARKER or INJURYMARKER nucleotide sequence shown in RISKMARKER or INJURYMARKER 1, and RISKMARKER or INJURYMARKER 6-8, 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 RISKMARKER or INJURYMARKER polypeptide may exist within a population (e.g., the human population). Such genetic polymorphism in the RISKMARKER or INJURYMARKER 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 RISKMARKER or INJURYMARKER protein, preferably a mammalian RISKMARKER or INJURYMARKER protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the RISKMARKER or INJURYMARKER gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in RISKMARKER or INJURYMARKER that are the result of natural allelic variation and that do not alter the functional activity of RISKMARKER or INJURYMARKER are intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding RISKMARKER or INJURYMARKER proteins from other species, and thus that have a nucleotide sequence that differs from the human sequence of RISKMARKER OR INJURYMARKER, are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the RISKMARKER or INJURYMARKER DNAs of the invention can be isolated based on their homology to the human RISKMARKER or INJURYMARKER 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 RISKMARKER or INJURYMARKER DNA can be isolated based on its homology to human membrane-bound RISKMARKER or INJURYMARKER. Likewise, a membrane-bound human RISKMARKER or INJURYMARKER DNA can be isolated based on its homology to soluble human RISKMARKER or INJURYMARKER.
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 RISKMARKER 1, or RISKMARKER 6-8. 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 RISKMARKER or INJURYMARKER 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.
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 formarmide.
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 6×SSC, 50 mM Tris-HCl (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 0.2×SSC, 0.01% BSA at 50° C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of RISKMARKER 1, or RISKMARKER 6-8 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 RISKMARKER 1, or RISKMARKER 6-8, 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 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55° C., followed by one or more washes in 1×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 RISKMARKER 1, or RISKMARKER 6-8, or 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% formarmide, 5×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 2×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 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 RISKMARKER sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced into an RISKMARKER nucleic acid or directly into an RISKMARKER polypeptide sequence without altering the functional ability of the RISKMARKER protein. In some embodiments, the nucleotide sequence of RISKMARKER 1, or RISKMARKER 6-8 will be altered, thereby leading to changes in the amino acid sequence of the encoded RISKMARKER 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 RISKMARKER 1, or RISKMARKER 6-8. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of RISKMARKER 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 RISKMARKER 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 RISKMARKER 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 RISKMARKER 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 RISKMARKER proteins that contain changes in amino acid residues that are not essential for activity. Such RISKMARKER proteins differ in amino acid sequence from the amino acid sequences of polypeptides encoded by nucleic acids containing RISKMARKER 1, or RISKMARKER 6-8, 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 the amino acid sequences of polypeptides encoded by nucleic acids comprising RISKMARKER 1, or RISKMARKER 6-8.
An isolated nucleic acid molecule encoding a RISKMARKER 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 RISKMARKER 1, or RISKMARKER 6-8, 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 RISKMARKER 1, or RISKMARKER 6-8 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 RISKMARKER 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 RISKMARKER coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for RISKMARKER biological activity to identify mutants that retain activity. Following mutagenesis of the nucleic acid, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.
In one embodiment, a mutant RISKMARKER protein can be assayed for (1) the ability to form protein:protein interactions with other RISKMARKER proteins, other cell-surface proteins, or biologically active portions thereof, (2) complex formation between a mutant RISKMARKER protein and a RISKMARKER ligand; (3) the ability of a mutant RISKMARKER 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 RISKMARKER protein antibody.
In other embodiment, the fragment of the complementary polynucleotide sequence described in claim 1 wherein the fragment of the complementary polynucleotide sequence hybridizes to the first sequence.
In other specific embodiments, the nucleic acid is RNA or DNA. The fragment or the fragment of the complementary polynucleotide sequence described in claim 38, wherein the fragment is between about 10 and about 100 nucleotides in length, e.g., between about 10 and about 90 nucleotides in length, or about 10 and about 75 nucleotides in length, about 10 and about 50 bases in length, about 10 and about 40 bases in length, or about 15 and about 30 bases in length.
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 RISKMARKER or INJURYMARKER 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, 50, 100, 250 or 500 nucleotides or an entire RISKMARKER or INJURYMARKER coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a RISKMARKER or INJURYMARKER protein, or antisense nucleic acids complementary to a nucleic acid comprising a RISKMARKER or INJURYMARKER 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 RISKMARKER or INJURYMARKER. 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 RISKMARKER. 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 RISKMARKER or INJURYMARKER 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 RISKMARKER or INJURYMARKER mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of RISKMARKER or INJURYMARKER mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of RISKMARKER or INJURYMARKER 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 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, 5-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, 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 in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a RISKMARKER or INJURYMARKER 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 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 α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual α-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 (Inoue et al. (1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA -DNA analogue (Inoue 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 RISKMARKER or INJURYMARKER mRNA transcripts to thereby inhibit translation of RISKMARKER or INJURYMARKER mRNA. A ribozyme having specificity for a RISKMARKER or INJURYMARKER -encoding nucleic acid can be designed based upon the nucleotide sequence of a RISKMARKER or INJURYMARKER 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 RISKMARKER or INJURYMARKER-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, RISKMARKER or INJURYMARKER 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, RISKMARKER or INJURYMARKER gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of a RISKMARKER or INJURYMARKER nucleic acid (e.g., the RISKMARKER or INJURYMARKER promoter and/or enhancers) to form triple helical structures that prevent transcription of the RISKMARKER or INJURYMARKER 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 RISKMARKER or INJURYMARKER 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 Chem 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; Perry-O'Keefe et al. (1996) PNAS 93: 14670-675.
PNAs of RISKMARKER or INJURYMARKER 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 RISKMARKER or INJURYMARKER 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., S1 nucleases (Hyrup B. (1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al (1996), above; Perry-O'Keefe (1996), above).
In another embodiment, PNAs of RISKMARKER or INJURYMARKER 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 RISKMARKER or INJURYMARKER 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 terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and 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) Nucl 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.
Riskmarker and Injurymarker Polypeptides
One aspect of the invention pertains to isolated RISKMARKER or INJURYMARKER 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-RISKMARKER or INJURYMARKER antibodies, e.g. antibodies against RISKMARKER 1, or RISKMARKER 6-8. In one embodiment, native RISKMARKER or INJURYMARKER proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, RISKMARKER or INJURYMARKER proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a RISKMARKER or INJURYMARKER 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 RISKMARKER or INJURYMARKER protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of RISKMARKER or INJURYMARKER 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 RISKMARKER or INJURYMARKER protein having less than about 30% (by dry weight) of non-RISKMARKER or INJURYMARKER protein (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-RISKMARKER or INJURYMARKER protein, still more preferably less than about 10% of non-RISKMARKER or INJURYMARKER protein, and most preferably less than about 5% non-RISKMARKER or INJURYMARKER protein. When the RISKMARKER or INJURYMARKER protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, ie., 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 RISKMARKER or INJURYMARKER 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 RISKMARKER or INJURYMARKER protein having less than about 30% (by dry weight) of chemical precursors or non-RISKMARKER or INJURYMARKER chemicals, more preferably less than about 20% chemical precursors or non-RISKMARKER or INJURYMARKER chemicals, still more preferably less than about 10% chemical precursors or non-RISKMARKER or INJURYMARKER chemicals, and most preferably less than about 5% chemical precursors or non-RISKMARKER or INJURYMARKER chemicals.
Biologically active portions of a RISKMARKER or INJURYMARKER protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the RISKMARKER or INJURYMARKER protein, e.g., the amino acid sequence encoded by a nucleic acid comprising RISKMARKER or INJURYMARKER 1-20 that include fewer amino acids than the full length RISKMARKER or INJURYMARKER proteins, and exhibit at least one activity of a RISKMARKER or INJURYMARKER protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the RISKMARKER or INJURYMARKER protein. A biologically active portion of a RISKMARKER or INJURYMARKER protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
A biologically active portion of a RISKMARKER or INJURYMARKER protein of the present invention may contain at least one of the above-identified domains conserved between the RISKMARKER or INJURYMARKER proteins. An alternative biologically active portion of a RISKMARKER or INJURYMARKER protein may contain at least two of the above-identified domains. Another biologically active portion of a RISKMARKER or INJURYMARKER protein may contain at least three of the above-identified domains. Yet another biologically active portion of a RISKMARKER or INJURYMARKER 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 RISKMARKER or INJURYMARKER protein.
In some embodiments, the RISKMARKER or INJURYMARKER protein is substantially homologous to one of these RISKMARKER or INJURYMARKER 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.
Determining Homology Between Two or More 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 Needleman and Wunsch 1970 J Mol 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 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 RISKMARKER 1, or RISKMARKER 6-8.
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 RISKMARKER chimeric or fusion proteins. As used herein, an RISKMARKER “chimeric protein” or “fusion protein” comprises an RISKMARKER polypeptide operatively linked to a non-RISKMARKER polypeptide. A “RISKMARKER polypeptide” refers to a polypeptide having an amino acid sequence corresponding to RISKMARKER, whereas a “non-RISKMARKER polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the RISKMARKER protein, e.g., a protein that is different from the RISKMARKER protein and that is derived from the same or a different organism. Within an RISKMARKER fusion protein the RISKMARKER polypeptide can correspond to all or a portion of an RISKMARKER protein. In one embodiment, an RISKMARKER fusion protein comprises at least one biologically active portion of an RISKMARKER protein. In another embodiment, an RISKMARKER fusion protein comprises at least two biologically active portions of an RISKMARKER protein. In yet another embodiment, an RISKMARKER fusion protein comprises at least three biologically active portions of an RISKMARKER protein. Within the fusion protein, the term “operatively linked” is intended to indicate that the RISKMARKER polypeptide and the non-RISKMARKER polypeptide are fused in-frame to each other. The non-RISKMARKER polypeptide can be fused to the N-terminus or C-terminus of the RISKMARKER polypeptide.
For example, in one embodiment an RISKMARKER fusion protein comprises an RISKMARKER 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 RISKMARKER activity (such assays are described in detail below).
In yet another embodiment, the fusion protein is a GST-RISKMARKER fusion protein in which the RISKMARKER 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 RISKMARKER, e.g. RISKMARKER 1, or RISKMARKER 6-8.
In another embodiment, the fusion protein is an RISKMARKER protein containing a heterologous signal sequence at its N-terminus. For example, a native RISKMARKER 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 RISKMARKER can be increased through use of a heterologous signal sequence.
In yet another embodiment, the fusion protein is a RISKMARKER-immunoglobulin fusion protein in which the RISKMARKER sequences comprising one or more domains are fused to sequences derived from a member of the immunoglobulin protein family. The RISKMARKER-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a RISKMARKER ligand and a RISKMARKER protein on the surface of a cell, to thereby suppress RISKMARKER-mediated signal transduction in vivo. The RISKMARKER-immunoglobulin fusion proteins can be used to affect the bioavailability of an RISKMARKER cognate ligand. Inhibition of the RISKMARKER ligand/RISKMARKER 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 RISKMARKER-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-RISKMARKER antibodies in a subject, to purify RISKMARKER ligands, and in screening assays to identify molecules that inhibit the interaction of RISKMARKER with a RISKMARKER ligand.
An RISKMARKER 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 carried 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). A RISKMARKER -encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the RISKMARKER protein.
Riskmarker and Injurymarker Agonists and Antagonists
The present invention also pertains to variants of the RISKMARKER or INJURYMARKER proteins that function as either RISKMARKER or INJURYMARKER agonists (mimetics) or as RISKMARKER or INJURYMARKER antagonists. Variants of the RISKMARKER or INJURYMARKER protein can be generated by mutagenesis, e.g., discrete point mutation or truncation of the RISKMARKER or INJURYMARKER protein. An agonist of the RISKMARKER or INJURYMARKER protein can retain substantially the same, or a subset of, the biological activities of the naturally occurring form of the RISKMARKER or INJURYMARKER protein. An antagonist of the RISKMARKER or INJURYMARKER protein can inhibit one or more of the activities of the naturally occurring form of the RISKMARKER or INJURYMARKER protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the RISKMARKER or INJURYMARKER 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 RISKMARKER or INJURYMARKER proteins.
Variants of the RISKMARKER or INJURYMARKER protein that function as either RISKMARKER or INJURYMARKER agonists (mimetics) or as RISKMARKER or INJURYMARKER antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the RISKMARKER or INJURYMARKER protein for RISKMARKER or INJURYMARKER protein agonist or antagonist activity. In one embodiment, a variegated library of RISKMARKER or INJURYMARKER variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of RISKMARKER or INJURYMARKER variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential RISKMARKER or INJURYMARKER sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of RISKMARKER or INJURYMARKER sequences therein. There are a variety of methods which can be used to produce libraries of potential RISKMARKER or INJURYMARKER variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene 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 RISKMARKER or INJURYMARKER sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu Rev Biochem 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 RISKMARKER or INJURYMARKER protein coding sequence can be used to generate a variegated population of RISKMARKER or INJURYMARKER fragments for screening and subsequent selection of variants of an RISKMARKER or INJURYMARKER protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a RISKMARKER or INJURYMARKER 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 S1 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 RISKMARKER or INJURYMARKER 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 gene libraries generated by the combinatorial mutagenesis of RISKMARKER or INJURYMARKER 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 RISKMARKER or INJURYMARKER variants (Arkin and Yourvan (1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6:327-331).
Anti-Riskmarker and Anti-Injurymarker Antibodies
An isolated RISKMARKER or INJURYMARKER protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind RISKMARKER or INJURYMARKER using standard techniques for polyclonal and monoclonal antibody preparation. The full-length RISKMARKER or INJURYMARKER protein can be used or, alternatively, the invention provides antigenic peptide fragments of RISKMARKER or INJURYMARKER for use as immunogens. The antigenic peptide of RISKMARKER or INJURYMARKER comprises at least 8 amino acid residues of the amino acid sequence encoded by a nucleic acid comprising the nucleic acid sequence shown in RISKMARKER 1-8 and INJURYMARKER 1-10 and encompasses an epitope of RISKMARKER or INJURYMARKER such that an antibody raised against the peptide forms a specific immune complex with RISKMARKER or INJURYMARKER. 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 RISKMARKER or INJURYMARKER 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.
RISKMARKER or INJURYMARKER polypeptides or derivatives, fragments, analogs or homologs thereof, may be utilized as immunogens in the generation of antibodies that immunospecifically-bind these 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, Fab and F(ab′)2 fragments, and an Fab expression library. Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies to an RISKMARKER or INJURYMARKER protein sequence, e.g. RISKMARKER 1 or RISKMARKER 6-8, 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 RISKMARKER or INJURYMARKER protein or a chemically synthesized RISKMARKER or INJURYMARKER 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 Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents. If desired, the antibody molecules directed against RISKMARKER or INJURYMARKER 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 RISKMARKER or INJURYMARKER. A monoclonal antibody composition thus typically displays a single binding affinity for a particular RISKMARKER or INJURYMARKER protein with which it immunoreacts. For preparation of monoclonal antibodies directed towards a particular RISKMARKER or INJURYMARKER 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 Nature 256: 495-497); the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: 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 RISKMARKER or INJURYMARKER protein (see e.g., U.S. Pat. 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 Fab fragments with the desired specificity for a RISKMARKER or INJURYMARKER 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. Pat. No. 5,225,539. Antibody fragments that contain the idiotypes to a RISKMARKER or INJURYMARKER protein may be produced by techniques known in the art including, but not limited to: (i) an F(ab′)2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab′2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) Fv fragments.
Additionally, recombinant anti-RISKMARKER or INJURYMARKER 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 al.(1988) Science 240:1041-1043; Liu et al. (1987) PNAS 84:3439-3443; Liu et al. (1987) J Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al. (1987) Cancer Res 47:999-1005; Wood et al. (1985) Nature 314:446449; Shaw et al. (1988) J Natl Cancer Inst. 80:1553-1559); Morrison(1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J Immunol 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 RISKMARKER or INJURYMARKER protein is facilitated by generation of hybridomas that bind to the fragment of a RISKMARKER or INJURYMARKER protein possessing such a domain. Antibodies that are specific for one or more domains within a RISKMARKER or INJURYMARKER protein, e.g., domains spanning the above-identified conserved regions of RISKMARKER or INJURYMARKER family proteins, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
Anti-RISKMARKER or anti-INJURYMARKER antibodies may be used in methods known within the art relating to the localization and/or quantitation of a RISKMARKER or INJURYMARKER protein (e.g., for use in measuring levels of the RISKMARKER or INJURYMARKER 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 RISKMARKER or INJURYMARKER 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-RISKMARKER or INJURYMARKER antibody (e.g., monoclonal antibody) can be used to isolate RISKMARKER or INJURYMARKER by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-RISKMARKER or INJURYMARKER antibody can facilitate the purification of natural RISKMARKER or INJURYMARKER from cells and of recombinantly produced RISKMARKER or INJURYMARKER expressed in host cells. Moreover, an anti-RISKMARKER or INJURYMARKER antibody can be used to detect RISKMARKER or INJURYMARKER protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the RISKMARKER or INJURYMARKER protein. Anti-RISKMARKER or INJURYMARKER 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 (ie., 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, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group 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 125I, 131 I, 35S or 3H.
Riskmarker Recombinant Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding RISKMARKER protein, e.g. RISKMARKER 1, or RISKMARKER 6-8, 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 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 intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) 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, Calif. (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., RISKMARKER proteins, mutant forms, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for expression of RISKMARKER in prokaryotic or eukaryotic cells. For example, RISKMARKER 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, Calif. (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 out 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 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 pRIT5 (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 11d (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, Calif. (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 RISKMARKER expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec 1 (Baldari, et al., (1987) EMBO J6:229-234), 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, Calif).
Alternatively, RISKMARKER 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 (Kaufmnan et al. (1987) EMBO J 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 Immunol 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;, 66). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the □-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 RISKMARKER 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 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, RISKMARKER 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 resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding RISKMARKER 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 RISKMARKER protein, e.g. RISKMARKER 1, or RISKMARKER 6-8. Accordingly, the invention further provides methods for producing RISKMARKER 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 RISKMARKER has been introduced) in a suitable medium such that RISKMARKER protein is produced. In another embodiment, the method further comprises isolating RISKMARKER from the medium or the host cell.
Pharmaceutical Compositions
The RISKMARKER nucleic acid molecules, RISKMARKER proteins, and anti-RISKMARKER or anti-INJURYMARKER 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.
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 bisulfite; 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.
Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a RISKMARKER protein or anti-RISKMARKER or INJURYMARKER 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 carrier. 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 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 carrier. 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, 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 Nucelic Acid Collections for Identifying Riskmarker and Injurymarker 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 RISKMARKER or INJURYMARKER sequences. In preferred embodiments, the kit includes reagents which detect 3, 4, 5, 6, 8, 10, 12, 15, or all of the RISKMARKER or INJURYMARKER nucleic acid sequences.
The invention also includes an isolated plurality of sequences which can identify one or more RISKMARKER or INJURYMARKER responsive nucleic acid sequences. The kit or plurality may include, e.g., sequence homologous to RISKMARKER or INJURYMARKER nucleic acid sequences, or sequences which can specifically identify one or more RISKMARKER or INJURYMARKER 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 to 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.

## Claims

1. An isolated nucleic acid consisting of the nucleic acid sequence of SEQ ID NO: 1, or its complement.

2. An isolated nucleic acid consisting of the nucleic acid sequence of SEQ ID NO: 2, or its complement.

3. An isolated nucleic acid consisting of the nucleic acid sequence of SEQ ID NO: 15, or its complement.

4. An isolated nucleic acid consisting of the nucleic acid sequence of SEQ ID NO: 17, or its complement.

5. A composition comprising the nucleic acid of claim 1.

6. A vector comprising the nucleic acid of claim 2.

7. A cell comprising the vector of claim 6.

8. A composition comprising the nucleic acid of claim 2.

9. A vector comprising the nucleic acid of claim 3.

10. A cell comprising the vector of claim 9.

11. A composition comprising the nucleic acid of claim 3.

12. A vector comprising the nucleic acid of claim 4.

13. A cell comprising the vector of claim 12.

14. A composition comprising the nucleic acid of claim 4.