CN101903774A - Prediction of genotoxicity - Google Patents

Abstract

The likelihood that a compound will exhibit genotoxicity in a micronucleus test is predicted by the ability of the compound to inhibit at least five kinases from a selected group.

Description

Predicted gene toxicity

The present invention relates generally to the toxicology field.More specifically, the present invention relates to the method for predicted gene toxicity and the method for screening latent gene toxic chemical.

Micronucleus test (" MNT ") is the common experiment of the conventional detection chromosome damage that uses in the pharmacy industry.Form micronucleus when complete chromosome or chromosome segment are not integrated into daughter nucleus after the silk division is finished.Cause the former and clastogen of the non-multiple of compound-cause of chromosome diminution/acquisition and fracture to make micronucleus form remarkable increase respectively, therefore can use this experiment to detect.Therefore, micronucleus is the biological marker of chromosome damage, and micronucleus test is to detect the sensitive method that causes former compound of non-multiple and/or clastogen compound.Micronucleus test is widely used as the evidence of genotoxicity (or not having genotoxicity) in pharmacy industry.

Yet, carry out micronucleus test effort and consuming time, when testing with cell toxic amount also false positive results can appear, and carrying out this test simultaneously also needs a large amount of article (cell, clone are kept reagent and compound).

Kinases is responsible for phosphorylated substrate and is transmitted intercellular and intracellular signal, and these signals are included in that chromosome replication in the mitosis process is initial, multiplication and stopping.Because known many signal cascades have effect in multiple disease, the frequent target of drugmaker suppresses kinases.Often exploitation micromolecule inhibitors of kinases (SMKI) comes competitiveness in conjunction with kinases ATP binding pocket, the ability of retardance enzyme phosphorylated substrate.Because ATP binding pocket high conservative in the kinases group, SMKI also often suppresses many kinases except that suppressing desired target, therefore should be noted that to follow the type medical compounds and the toxicity of next non-target kinase inhibition.More specifically, confirmed that the genetoxic after metaphase is the common toxicity tendency of SMKI as positive micronucleus result.

Now, very fast by using, reagent dosage is less, be easy to automated method, we have invented the method which kind of compound in the prediction micronucleus test will present the positive (being genotoxicity) result.

By detecting the interaction (kinases combination and/or inhibition) between given compound and a large amount of kinases, the invention provides the given compound of fast measuring will represent the possibility of genotoxicity in the MNT experiment method.Because can fast and use automated process to measure kinase inhibition and/or combination, method of the present invention can be at genotoxicity (or not having genotoxicity) high flux screening compound.

In fact, can use means known in the art to measure combination and inhibition.For example referring to M.A.Fabian etc., Nature Biotechnol (2005) 23:329-36 (introducing here as a reference) with its full content.

One aspect of the present invention is the method for predictive compound genotoxicity, and this method comprises step: test compounds is provided; Measure the ability that this compound suppresses at least 10 kinds of kinase whose kinase activities, described kinases is selected from by CDK2 (Seq.Id.1), CLK1 (Seq.Id.2), DYRK1B (Seq.Id.3), ERK8 (Seq.Id.4), GSK3A (Seq.Id.5), GSK3B (Seq.Id.6), PCTK1 (Seq.Id.7), PCTK2 (Seq.Id.8), STK16 (Seq.Id.9), TTK (Seq.Id.10), CLK2 (Seq.Id.11), the group that ERK3 (Seq.Id.12) and PRKR (Seq.Id.13) form, or be selected from by CDK2, CLK1, DYRK1B, ERK8 (MAPK15), GSK3A, GSK3B, PCTK1, PCTK2, STK16, TTK, CDK7 (Seq.Id.64), the group that CLK4 (Seq.Id.68) and PCTK3 (Seq.Id.69) form, wherein at least 5 kinds of described kinases are suppressed at least 50% activity and show that described test compounds may have genotoxicity.

In one embodiment, with the described test compounds of about 10 μ M concentration determinations.

In another embodiment, tested the ability that compound suppresses to be selected from least 12 kinds of kinase whose kinase activities of described group.In another embodiment, measured the ability that compound suppresses whole 13 kinds of kinase whose kinase activities in described group.

In another embodiment, except above-mentioned 13 kinds kinase whose group, also can test at least a other following kinases.Compound is relevant with higher possible genotoxicity with one or more high affinities of planting these other kinases (except 13 kinds of kinases that great majority have been identified).These other kinases are: MKNK2 (Seq.Id.14), SgK085 (Seq.Id.15), PIM2 (Seq.Id.16), TNNI3K (Seq.Id.17), KIT (Seq.Id.18), MELK (Seq.Id.19), AURKA (Seq.Id.20), CLK3 (Seq.Id.21), AAK1 (Seq.Id.22), DCAMKL3 (Seq.Id.23), LIMK1 (Seq.Id.24), FLT1 (Seq.Id.25), MAP2K4 (Seq.Id.26), PIM3 (Seq.Id.27), AURKB (Seq.Id.28), ERK2 (Seq.Id.29), CSNK1A1L (Seq.Id.30), DAPK3 (Seq.Id.31), MLCK (Seq.Id.32), CLK3 (Seq.Id.33), PFTK1 (Seq.Id.34), PRKD3 (Seq.Id.35), AURKC (Seq.Id.36), ERK5 (Seq.Id.37), STK17A (Seq.Id.38), MST4 (Seq.Id.39), CDK3 (Seq.Id.40), MYLK (Seq.Id.41), CDC2L1 (Seq.Id.42), QIK (Seq.Id.43), CDK11 (Seq.Id.44), PLK1 (Seq.Id.45), PDGFR β (Seq.Id.46), PRKCM (Seq.Id.47), MAPK4 (Seq.Id.48), PIP5K2B (Seq.Id.49), CSNK1D (Seq.Id.50), RPS6KA1 (KD1) (Seq.Id.51), CDK5 (Seq.Id.52), PLK3 (Seq.Id.53), BIKE (Seq.Id.54), PLK4 (Seq.Id.55), CAMK2A (Seq.Id.56), STK3 (Seq.Id.57), CSNK2A1 (Seq.Id.58), STK17B (Seq.Id.59), CDK8 (Seq.Id.60), MAP2K6 (Seq.Id.61), PIM1 (Seq.Id.62), MAP2K3 (Seq.Id.63), CDK7 (Seq.Id.64), IKK 8 (Seq.Id.65), TGFBR2 (Seq.Id.66), CDK9 (Seq.Id.67), CLK4 (Seq.Id.68) and PCTK3 (Seq.Id.69).

Another aspect of the present invention is the method for screening candidate latent gene toxic chemical, and the method comprising the steps of: multiple compound is provided; Measure the ability that each compound suppresses multiple kinase whose kinase activity, described kinases is selected from by CDK2, CLK1, DYRK1B, ERK8, GSK3A, GSK3B, PCTK1, PCTK2, STK16, TTK, CLK2, the group that ERK3 and PRKR form, or optional free CDK2, CLK1, DYRK1B, ERK8 (MAPK15), GSK3A, GSK3B, PCTK1, PCTK2, STK16, TTK, CDK7, the group that CLK4 and PCTK3 form, at least 50% or the specificity that wherein suppress at least 5 kinds of described kinase activities show that in conjunction with at least 5 kinds described kinase whose at least 50% described test compounds may have genotoxicity.In preferred embodiments, this method further comprises the step of compound that refusal has the genotoxicity of possibility.

Generally speaking, compound is closely related with the ability that given kinase whose binding affinity and this compound suppress this kinase activity, so binding affinity is the reliable substitute of the activity of inhibition.Therefore, in preferred embodiments, the ability that compound suppresses kinase activity is by measuring this compound and described kinase whose binding affinity is measured.Can use several different methods known in the art to measure binding affinity; For example measure binding affinity by the competitive assay that uses fixing change kinases (or fixing test compounds, or fixing competitive part, any of these material also can be labeled).Can use standard method fixed compound and kinases, for example fix with being captured on the substrate that is coated with streptavidin by biotinylation.

Therefore, people can preparation example as having the kinase whose test substrate of various fixed, preferably comprise 13 kinds of kinases: CDK2, the CLK1, DYRK1B, ERK8 (MAPK15), GSK3A, GSK3B, PCTK1, PCTK2, STK16, TTK, CLK2, ERK3 and the PRKR (or kinases of other evaluations) that identify herein.Can be with kinases directly (promptly by absorption, covalent bond or biotin-avidin combination, Deng) or indirectly (for example be fixed on lip-deep part by being attached to, described fixing by absorption, covalent bond, biotin-avidin in conjunction with or other connections realize) be fixed to the surface.Then, kinases is contacted and measure affinity (or enzymatic activity inhibition) with test compounds, for example measure affinity through tagged compound or loss through mark competition thing by what measure combination.

Measure every kind of compound at least 10 kinds through the kinase whose kinases affinity through identifying that identify or selectable, and most preferably at the kinases of whole 13 kinds of evaluations: the genotoxicity prediction of using the kinases (13 kinds at the most) of greater number to produce has high confidence.Compound with high gross activity (for example show and have at least 5 kinds of kinases in 13 kinds of kinases preferred 8 kinds or more kinds of kinase whose high affinity) has the genotoxicity of high likelihood: predict that this compound is positive in the test of MNT genotoxicity.The compound that prediction has a low gross activity (for example only the kinases of identifying is had low-affinity or the kinases only the 1-4 kind identified has high affinity) is tested in MNT and is negative.

Generally will in mensuration of the present invention, be accredited as " genotoxicity " or " latent gene toxicity " for the drug candidate (promptly predict and in MNT, be the medicine of genotoxicity) of positive test, and refusal falls or cancels from further exploitation.Under the situation that high flux screening is used, such compound can be labeled as " toxicity " (for example, under the situation of robotization high throughput system, realizing) by the software of management system, therefore can make decision as early as possible.

Therefore, part is based on the latent gene toxicity of compound, and people can use method of the present invention to distinguish priority ranking and select candidate's the compound that is used for drug development.For example, be the further Asia collection of distinguishing priority ranking or selecting described compound of developing if someone has prepared many compound (for example 50 kinds or more) and expectations that have at the similar activity of selecting target, he can test whole group compound in the method for the invention and lose or refuse the compound of all that genotoxicity positive test.Reduced the expense of drug development by the important source of early stage evaluation toxicity, and the research quantity of any compound of developing of choosing.Because method of the present invention is quick and be easy to robotization, the screening of huge amount compound becomes possibility now, otherwise it can not or not gear to actual circumstances.

Also can use method of the present invention to identify environmental contaminants etc., in this case, identify that typically such compound is with their toxicity of further research.In this application of the inventive method, people can use known method (for example chromatography) classification environmental sample (for example suspecting contaminated soil, water or air), and study described component with method of the present invention.Then, the component that shows the genotoxicity signal is carried out further classification, and (using method of the present invention) identifies the toxic agents that works.Alternatively, people can use suspection to carry out method of the present invention as the pure compound of environmental contaminants or the compound of purifying, to measure its potential genotoxicity.Because method of the present invention is quick and be easy to robotization, the screening of huge amount compound becomes possibility now, otherwise it can not or not gear to actual circumstances.

All publication documents of quoting in this open text are with its whole introducing here as a reference.

Except as otherwise noted, the following term of using in the application's (comprising instructions and claim) provides and is defined as follows.It must be noted that unless context offers some clarification in addition, " a " of used singulative, " an " reach " the " and comprise plural object in instructions and accessory claim book.

Term " genotoxicity " refers to produce the compound of chromosome aberration as used herein, comprises fracture (clastogen) or unusual copy number (it is former to cause non-multiple).In this context, " genotoxicity " refers to the positive findings in the micronucleus test." possibility of genotoxicity " refers to that specifically the compound of forecasting institute discussion proves genotoxicity with at least 75% degree of confidence in MNT.

Term " test compounds " refers to measure the material of its genotoxicity.Test compounds can be drug candidate or lead compound, chemical intermediate, environmental contaminants, compound mixture etc.

Term " kinases " refers to add to protein or molecular phosphorus acidic group and/or removes the enzyme of phosphate from protein or molecule." inhibition kinase activity " refers to that compound reduces or disturb the ability of such phosphatase activity.Because the ability of micromolecule and given kinase whose binding affinity and described molecules in inhibiting kinase activity is closely related, think that binding affinity is the synonym of kinase activity described herein, thinks that high binding affinity is equal to high kinase inhibiting activity.Correlativity between binding affinity and kinase inhibition is by M.A.Fabian etc., and Nature Biotechnol (2005) 23:329-36 (introducing herein as a reference in full) describes.

Term " kinases of evaluation " refers to following kinase whose collection: CDK2 (Seq.Id.1), CLK1 (Seq.Id.2), DYRK1B (Seq.Id.3), ERK8 (Seq.Id.4), GSK3A (Seq.Id.5), GSK3B (Seq.Id.6), PCTK1 (Seq.Id.7), PCTK2 (Seq.Id.8), STK16 (Seq.Id.9), TTK (Seq.Id.10), CLK2 (Seq.Id.11), ERK3 (Seq.Id.12) and PRKR (Seq.Id.13)." kinases of other evaluations " refer to the kinases collection be made up of CDK2, CLK1, DYRK1B, ERK8 (MAPK15), GSK3A, GSK3B, PCTK1, PCTK2, STK16, TTK, CDK7, CLK4 and PCTK3.Preferred kinases is the human kinase of indicating in the sequence table.Yet, also can use in the method to derive from any other biological kinases.

All patents and the publication addressed are all incorporated this paper into as a reference with its integral body herein.

Embodiment

In order to identify that the indication test compounds is the kinases collection of the possibility of genotoxicity, has carried out following analysis.At first, select 54 kinds of suitable micromolecule inhibitors of kinases (" SMKI ") to form training set.Secondly, each compound in the training set all need obtain to suppress spectrum (single point inhibition profile) at 290 kinds of kinase whose external MNT results and single-point.Carry out statistical study then, set up the model that uses described single-point kinase inhibition spectrum with (1) and identify the kinases relevant with MNT result to predict described MNT result and (2).At last, the collection that is not used to other 33 kinds of SMKI of training is verified this model.

External micronucleus test (M.Fenech, Mutation Res (2000) 455 (1-2): 81-95) had before been described in detail.It is L5178Y tk that the immortal mouse lymphoma cell of the suspension growth of having set up has been used in this experiment ^+/-(ATCC CRL 9518).Usually, with at least 12 concentration levels and be up to the concentration determination compound of 500 μ g/mL.The highest assessment dosage of general selection is observed tangible deposited phenomenon in acceptable toxicity (cell count (RCC) reduction is less than 50% relatively) or the aqueous medium.If this compound is solvable and the nontoxic maximum dose level of then setting 5000 μ g/mL.In order to assess cytotoxicity, calculate relative cell count (RCC represents with the % negative control).By setting cell density is about 1 * 10 ⁶Individual cell/mL and use cytospin (1000 rev/mins, 5 minutes) centrifugal to clean slide and the preparation slide.(20 ℃, at least 4 hours) fixing and storage cell in ice-cold methyl alcohol.With H33258 (1 μ g/mL PBS/CMF) hatch slide 5 minutes and with 10 μ L antifade mountings to be used for fluorescence microscopy.Under the help of the epifluorescence microscope that is equipped with suitable filter set, analyze minimum 3 concentration levels to determine existing of micronucleation cell.If compare with parallel negative control, one or more concentration has shown the micronucleation cell number of at least 2 times of increases, think that then this compound has clastogen/the cause former activity of non-multiple.

Based on a plurality of standards (comprising that selective kinase suppresses spectrum, minimization of redundancy and Chemical Diversity) 54 compounds are selected to be included in the training set.In inner SMKI database, only consider to have the compound that selective kinase suppresses spectrum, wherein alternative cpd is to suppress 6 kinds or the kinase whose inhibiting value of less kind greater than 95% at single-point, and suppresses 11 kinds or the kinase whose inhibiting value of less kind greater than 85% compound.Use M.A.Fabian etc., the described method of Nature Biotechnol (2005) 23:329-36 is measured kinase inhibition.When the anti-many identical kinases of a large amount of compound selectives, only select a kind of in these compounds, to minimize those kinase whose redundances or to repeat expressivity (over-representation).Behind these filtration steps, select the Chemical Diversity collection based on physical property (comprising AlogP, molecular weight, hydrogen donor and acceptor number, rotatable number of keys, atomicity, number of rings, fragrant number of rings and segments).In SciTegic ' s Pipeline Pilot 6.0.2, define diversity based on maximal phase opposite sex method and use " diversity molecule " filter (" Diverse Molecules " filter).

Obtain in the training set anti-290 kinds of kinase whose inhibition spectrums of every kind of compound and external MNT result (N=54).3 different readings have been obtained at MNT result: negative (N=22), positive (N=26) and the weak positive (N=6).Based on the %MN cell on the concentration that suppresses to compose the positive a little less than 6 is assigned as negative label or positive label.It is negative that this is reallocated in 6 kinds of compounds 5 kinds, provided 27 feminine genders and 27 positive compounds altogether.

In the scope of all inhibition spectrum collection, at first carry out pre-service, do not provide kinases information or bias to remove.To all there be the kinases of difference to remove concentrating of whole 54 kinds of compounds, because they do not provide information.JNK and p38 isotype are removed, to reduce the bias that exploitation comes a large amount of compounds in those kinase whose training sets of target.For guaranteeing to remove JNK and the p38 isotype is not introduced multi-form bias, we have carried out extra analysis, we have only considered that those are not developed and have been used for these kinase target target training set compounds whereby, and find that JNK and p38 isotype all have nothing to do with MNT result.

In order to set up this model, feature selecting (FS) and pattern-recognition (PR) have been carried out in a plurality of stages.Analyze for all, use cross validation to assess the performance of this model in a plurality of tests.Each test is divided into training set and test set at random with raw data; Use training set to set up temporary pattern, the use test collection predicts the outcome, check performance then.Which kind of kinases is measured in the use characteristic system of selection or " feature " may be the most relevant with MNT result.In each test, with the input value that resists the inhibiting value of selected feature as mode identification method, it has predicted the positive or negative result then.

In first stage, feature selection approach is divided into two groups: can handle the method (FS1) of a large amount of input data sets and the method for better carrying out with less data (FS2).Use 10 5 times of crosses to test, in a plurality of analysis of experimentss, tested the various combination of FS1, FS2 and PR.The method that selection has minimum average error rate makes up the analysis that is used for next stage.These combinations comprise that Ke's Er Monuofu-Vladimir Smirnov/T that is used for FSI tests hybrid algorithm, the random forest algorithm and the support vector machine (T.Hastie etc. that are used for PR that are used for FS2, " The Elements of StatisticalLearning " (2001, Springer-Verlag); R.O.Duda etc., and " Pattern Classification, second edition " (2000, Wiley-Interscience); " Feature Extraction-Foundationsand Applications " (2006, Springer-Verlag, editors such as I.Guyon)).

The method combination that to select the phase one is adjusted to obtain optimal representation.Optimize a plurality of parameters, comprised the kinases number that uses in the model.Adjustment process shows, in a plurality of tests, the average error rate is minimum when being chosen for significant kinases number being 13 behind FS1 and FS2.Therefore, adjusted this model with optimized parameter, 13 notable features of specific then selection are as the input value of PR.

Accuracy, characteristic number and the optimal adjusting parameters of the model that uses this feature selecting and mode identification method combination have been assessed by carrying out 50 5 times of cross validations then.Importantly, in each cross validation multiple, carry out feature selecting and pattern-recognition.The model that produces has 80% ± 4% accuracy: promptly, this model has on average correctly been predicted MNT result under 80% situation.

Also use 50 5 times of cross validations to measure the kinases relevant with MNT result.Be chosen as significant selection of times kinases based on (50 5 times of cross validations) kinases in 250 tests.At least once elected as significantly for 55 kinds in 290 kinds of original kinases.Selection is included in the final model with selecteed those kinases of frequency (N=13) greater than 50%.After many wheel tests, find that anti-these 13 kinds kinase whose kinase inhibition spectrums are significant under at least 50% the true MNT result's of prediction situation.That is, the SMKI with positive external MNT result tends to have high-caliber anti-these 13 kinds kinase whose inhibition activity.

For each SMKI, this model is made up of anti-following 13 kinds of kinase whose single-point kinase inhibition spectrums: CDK2, CLK1, DYRK1B, ERK8 (MAPK15), GSK3A, GSK3B, PCTK1, PCTK2, STK16, TTK, CLK2, ERK3 and PRKR.In addition, the external MNT measurement result that also comprises selected concentration when carrying out the kinases screening.13 kinds of kinases: CDK2, the CLK1, DYRK1B, ERK8 (MAPK15), GSK3A, GSK3B, PCTK1, PCTK2, STK16, TTK, CDK7, CLK4 and the PCTK3 that comprise second (overlapping) collection based on second model of quantitative binding constant.Two selected kinases height of model are similar, and this has proved the robustness of single-point kinase inhibition model.

In order to assess the validity of this final mask, 33 kinds of compounds that collect are in addition collected as checking.These 33 kinds of compounds are not included in concentrating of 54 kinds of initial compounds, but each compound includes anti-kinase whose single-point inhibiting value of described 13 kinds of models and external MNT result.If provided verification msg, this model can accurately be predicted the MNT result of all compounds, therefore carries out with 76% accuracy, and this accuracy is within our the model accuracy scope according to the cross validation estimation.

The present invention is by being described with reference to its specific embodiments, those skilled in the art will appreciate that can make some changes and can replace with equivalent under the situation that does not deviate from the spirit and scope of the present invention.In addition, can carry out multiple modification makes special environment, material, article composition, method, process steps or step be adapted to target spirit and scope of the present invention.All such modifications are defined as falling within the scope of this appending claims.

Sequence table

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agccaaagca?cccgacccga?agaaaagaaa?gacgaaggac?agccccgaag?agacacagac 1200

gacagcaaaa?aaagagacag?cgaaacaaca?aagccagagg?ggaaggcaaa?gacaagcaag 1260

gaagaacacc?agaagaaagg?aacagaaaaa?ggcagaagga?agacgaacag?gcaagaccac 1320

ccaccaaaag?aaggacaag 1339

 

<210>3

<211>629

<212>PRT

＜213〉people

 

<400>3

 

Met?Ala?Val?Pro?Pro?Gly?His?Gly?Pro?Phe?Ser?Gly?Phe?Pro?Gly?Pro

1 5 10 15

Gln?Glu?His?Thr?Gln?Val?Leu?Pro?Asp?Val?Arg?Leu?Leu?Pro?Arg?Arg

20 25 30

Leu?Pro?Leu?Ala?Phe?Arg?Asp?Ala?Thr?Ser?Ala?Pro?Leu?Arg?Lys?Leu

35 40 45

Ser?Val?Asp?Leu?Ile?Lys?Thr?Tyr?Lys?His?Ile?Asn?Glu?Val?Tyr?Tyr

50 55 60

Ala?Lys?Lys?Lys?Arg?Arg?Ala?Gln?Gln?Ala?Pro?Pro?Gln?Asp?Ser?Ser

65 70 75 80

Asn?Lys?Lys?Glu?Lys?Lys?Val?Leu?Asn?His?Gly?Tyr?Asp?Asp?Asp?Asn

85 90 95

His?Asp?Tyr?Ile?Val?Arg?Ser?Gly?Glu?Arg?Trp?Leu?Glu?Arg?Tyr?Glu

100 105 110

Ile?Asp?Ser?Leu?Ile?Gly?Lys?Gly?Ser?Phe?Gly?Gln?Val?Val?Lys?Ala

115 120 125

Tyr?Asp?His?Gln?Thr?Gln?Glu?Leu?Val?Ala?Ile?Lys?Ile?Ile?Lys?Asn

130 135 140

Lys?Lys?Ala?Phe?Leu?Asn?Gln?Ala?Gln?Ile?Glu?Leu?Arg?Leu?Leu?Glu

145 150 155 160

Leu?Met?Asn?Gln?His?Asp?Thr?Glu?Met?Lys?Tyr?Tyr?Ile?Val?His?Leu

165 170 175

Lys?Arg?His?Phe?Met?Phe?Arg?Asn?His?Leu?Cys?Leu?Val?Phe?Glu?Leu

180 185 190

Leu?Ser?Tyr?Asn?Leu?Tyr?Asp?Leu?Leu?Arg?Asn?Thr?His?Phe?Arg?Gly

195 200 205

Val?Ser?Leu?Asn?Leu?Thr?Arg?Lys?Leu?Ala?Gln?Gln?Leu?Cys?Thr?Ala

210 215 220

Leu?Leu?Phe?Leu?Ala?Thr?Pro?Glu?Leu?Ser?Ile?Ile?His?Cys?Asp?Leu

225 230 235 240

Lys?Pro?Glu?Asn?Ile?Leu?Leu?Cys?Asn?Pro?Lys?Arg?Ser?Ala?Ile?Lys

245 250 255

Ile?Val?Asp?Phe?Gly?Ser?Ser?Cys?Gln?Leu?Gly?Gln?Arg?Ile?Tyr?Gln

260 265 270

Tyr?Ile?Gln?Ser?Arg?Phe?Tyr?Arg?Ser?Pro?Glu?Val?Leu?Leu?Gly?Thr

275 280 285

Pro?Tyr?Asp?Leu?Ala?Ile?Asp?Met?Trp?Ser?Leu?Gly?Cys?Ile?Leu?Val

290 295 300

Glu?Met?His?Thr?Gly?Glu?Pro?Leu?Phe?Ser?Gly?Ser?Asn?Glu?Val?Asp

305 310 315 320

Gln?Met?Asn?Arg?Ile?Val?Glu?Val?Leu?Gly?Ile?Pro?Pro?Ala?Ala?Met

325 330 335

Leu?Asp?Gln?Ala?Pro?Lys?Ala?Arg?Lys?Tyr?Phe?Glu?Arg?Leu?Pro?Gly

340 345 350

Gly?Gly?Trp?Thr?Leu?Arg?Arg?Thr?Lys?Glu?Leu?Arg?Lys?Asp?Tyr?Gln

355 360 365

Gly?Pro?Gly?Thr?Arg?Arg?Leu?Gln?Glu?Val?Leu?Gly?Val?Gln?Thr?Gly

370 375 380

Gly?Pro?Gly?Gly?Arg?Arg?Ala?Gly?Glu?Pro?Gly?His?Ser?Pro?Ala?Asp

385 390 395 400

Tyr?Leu?Arg?Phe?Gln?Asp?Leu?Val?Leu?Arg?Met?Leu?Glu?Tyr?Glu?Pro

405 410 415

Ala?Ala?Arg?Ile?Ser?Pro?Leu?Gly?Ala?Leu?Gln?His?Gly?Phe?Phe?Arg

420 425 430

Arg?Thr?Ala?Asp?Glu?Ala?Thr?Asn?Thr?Gly?Pro?Ala?Gly?Ser?Ser?Ala

435 440 445

Ser?Thr?Ser?Pro?Ala?Pro?Leu?Asp?Thr?Cys?Pro?Ser?Ser?Ser?Thr?Ala

450 455 460

Ser?Ser?Ile?Ser?Ser?Ser?Gly?Gly?Ser?Ser?Gly?Ser?Ser?Ser?Asp?Asn

465 470 475 480

Arg?Thr?Tyr?Arg?Tyr?Ser?Asn?Arg?Tyr?Cys?Gly?Gly?Pro?Gly?Pro?Pro

485 490 495

Ile?Thr?Asp?Cys?Glu?Met?Asn?Ser?Pro?Gln?Val?Pro?Pro?Ser?Gln?Pro

500 505 510

Leu?Arg?Pro?Trp?Ala?Gly?Gly?Asp?Val?Pro?His?Lys?Thr?His?Gln?Ala

515 520 525

Pro?Ala?Ser?Ala?Ser?Ser?Leu?Pro?Gly?Thr?Gly?Ala?Gln?Leu?Pro?Pro

530 535 540

Gln?Pro?Arg?Tyr?Leu?Gly?Arg?Pro?Pro?Ser?Pro?Thr?Ser?Pro?Pro?Pro

545 550 555 560

Pro?Glu?Leu?Met?Asp?Val?Ser?Leu?Val?Gly?Gly?Pro?Ala?Asp?Cys?Ser

565 570 575

Pro?Pro?His?Pro?Ala?Pro?Ala?Pro?Gln?His?Pro?Ala?Ala?Ser?Ala?Leu

580 585 590

Arg?Thr?Arg?Met?Thr?Gly?Gly?Arg?Pro?Pro?Leu?Pro?Pro?Pro?Asp?Asp

595 600 605

Pro?Ala?Thr?Leu?Gly?Pro?His?Leu?Gly?Leu?Arg?Gly?Val?Pro?Gln?Ser

610 615 620

Thr?Ala?Ala?Ser?Ser

625

 

<210>4

<211>544

<212>PRT

＜213〉people

 

<400>4

 

Met?Cys?Thr?Val?Val?Asp?Pro?Arg?Ile?Val?Arg?Arg?Tyr?Leu?Leu?Arg

1 5 10 15

Arg?Gln?Leu?Gly?Gln?Gly?Ala?Tyr?Gly?Ile?Val?Trp?Lys?Ala?Val?Asp

20 25 30

Arg?Arg?Thr?Gly?Glu?Val?Val?Ala?Ile?Lys?Lys?Ile?Phe?Asp?Ala?Phe

35 40 45

Arg?Asp?Lys?Thr?Asp?Ala?Gln?Arg?Thr?Phe?Arg?Glu?Ile?Thr?Leu?Leu

50 55 60

Gln?Glu?Phe?Gly?Asp?His?Pro?Asn?Ile?Ile?Ser?Leu?Leu?Asp?Val?Ile

65 70 75 80

Arg?Ala?Glu?Asn?Asp?Arg?Asp?Ile?Tyr?Leu?Val?Phe?Glu?Phe?Met?Asp

85 90 95

Thr?Asp?Leu?Asn?Ala?Val?Ile?Arg?Lys?Gly?Gly?Leu?Leu?Gln?Asp?Val

100 105 110

His?Val?Arg?Ser?Ile?Phe?Tyr?Gln?Leu?Leu?Arg?Ala?Thr?Arg?Phe?Leu

115 120 125

His?Ser?Gly?His?Val?Val?His?Arg?Asp?Gln?Lys?Pro?Ser?Asn?Val?Leu

130 135 140

Leu?Asp?Ala?Asn?Cys?Thr?Val?Lys?Leu?Cys?Asp?Phe?Gly?Leu?Ala?Arg

145 150 155 160

Ser?Leu?Gly?Asp?Leu?Pro?Glu?Gly?Pro?Glu?Asp?Gln?Ala?Val?Thr?Glu

165 170 175

Tyr?Val?Ala?Thr?Arg?Trp?Tyr?Arg?Ala?Pro?Glu?Val?Leu?Leu?Ser?Ser

180 185 190

His?Arg?Tyr?Thr?Leu?Gly?Val?Asp?Met?Trp?Ser?Leu?Gly?Cys?Ile?Leu

195 200 205

Gly?Glu?Met?Leu?Arg?Gly?Arg?Pro?Leu?Phe?Pro?Gly?Thr?Ser?Thr?Leu

210 215 220

His?Gln?Leu?Glu?Leu?Ile?Leu?Glu?Thr?Ile?Pro?Pro?Pro?Ser?Glu?Glu

225 230 235 240

Asp?Leu?Leu?Ala?Leu?Gly?Ser?Gly?Cys?Arg?Ala?Ser?Val?Leu?His?Gln

245 250 255

Leu?Gly?Ser?Arg?Pro?Arg?Gln?Thr?Leu?Asp?Ala?Leu?Leu?Pro?Pro?Asp

260 265 270

Thr?Ser?Pro?Glu?Ala?Leu?Asp?Leu?Leu?Arg?Arg?Leu?Leu?Val?Phe?Ala

275 280 285

Pro?Asp?Lys?Arg?Leu?Ser?Ala?Thr?Gln?Ala?Leu?Gln?His?Pro?Tyr?Val

290 295 300

Gln?Arg?Phe?His?Cys?Pro?Ser?Asp?Glu?Trp?Ala?Arg?Glu?Ala?Asp?Val

305 310 315 320

Arg?Pro?Arg?Ala?His?Glu?Gly?Val?Gln?Leu?Ser?Val?Pro?Glu?Tyr?Arg

325 330 335

Ser?Arg?Val?Tyr?Gln?Met?Ile?Leu?Glu?Cys?Gly?Gly?Ser?Ser?Gly?Thr

340 345 350

Ser?Arg?Glu?Lys?Gly?Pro?Glu?Gly?Val?Ser?Pro?Ser?Gln?Ala?His?Leu

355 360 365

His?Lys?Pro?Arg?Ala?Asp?Pro?Gln?Leu?Pro?Ser?Arg?Thr?Pro?Val?Gln

370 375 380

Gly?Pro?Arg?Pro?Arg?Pro?Gln?Ser?Ser?Pro?Gly?His?Asp?Pro?Ala?Glu

385 390 395 400

His?Glu?Ser?Pro?Arg?Ala?Ala?Lys?Asn?Val?Pro?Arg?Gln?Asn?Ser?Ala

405 410 415

Pro?Leu?Leu?Gln?Thr?Ala?Leu?Leu?Gly?Asn?Gly?Glu?Arg?Pro?Pro?Gly

420 425 430

Ala?Lys?Glu?Ala?Pro?Pro?Leu?Thr?Leu?Ser?Leu?Val?Lys?Pro?Ser?Gly

435 440 445

Arg?Gly?Ala?Ala?Pro?Ser?Leu?Thr?Ser?Gln?Ala?Ala?Ala?Gln?Val?Ala

450 455 460

Asn?Gln?Ala?Leu?Ile?Arg?Gly?Asp?Trp?Asn?Arg?Gly?Gly?Gly?Val?Arg

465 470 475 480

Val?Ala?Ser?Val?Gln?Gln?Val?Pro?Pro?Arg?Leu?Pro?Pro?Glu?Ala?Arg

485 490 495

Pro?Gly?Arg?Arg?Met?Phe?Ser?Thr?Ser?Ala?Leu?Gln?Gly?Ala?Gln?Gly

500 505 510

Gly?Ala?Arg?Ala?Leu?Leu?Gly?Gly?Tyr?Ser?Gln?Ala?Tyr?Gly?Thr?Val

515 520 525

Cys?His?Ser?Ala?Leu?Gly?His?Leu?Pro?Leu?Leu?Glu?Gly?His?His?Val

530 535 540

 

<210>5

<211>1789

<212>RNA

＜213〉people

 

<220>

<221>misc_feature

<222>(3)..(3)

＜223〉n is a, c, g or u

 

<400>5

rgncccaagc?cagagcggcg?cggccggaag?aggccagggc?ccgggggagg?cggcggcagc 60

ggcggcggcg?gggcagcccg?ggcagcccga?gccccgcagc?cgggccggcc?ggcgccagag 120

cggcggcggg?cccgggaggc?ggcccggggg?ccgggcaggg?cgcggacagc?cgcgcggagc 180

ccggcggcgg?aggcggagga?ggcggcggcg?gccccggagg?ccggccccgg?cccaggcggc 240

accggcggcg?gaaaggcacg?cggggccagg?ggggggcgcg?gggcccgagc?ccggggggga 300

cccggcggca?gcggcggagg?aggcagcgga?ggccccggcg?caggcacagc?cccgccgccc 360

gggggaagcg?ggccggacag?cgggaaggga?ccacagcgag?ccaccaggcc?aaggcccaga 420

gcgccccaag?aagggcacac?ggacacaaag?gaggcaaggc?caggggcgga?ccaggcacgg 480

cggcagagac?cagggaacag?cgccacaaga?aggcccagga?caagaggcaa?gaaccgagag 540

cgcagacagc?gaagcggacc?acgcaaagga?ggcgagaacc?acccagggcg?agaagaaaga 600

cgagcaccaa?acgggcggaa?aggcccgaga?caggaccggg?ggcccgccac?caccaaggcc 660

aaggaccacc?cacccagcaa?gggacagacc?agccccgcag?cggccacacc?accccagggc 720

gggcaccgcg?acacaagccc?cagaaccgcg?gggacccgac?acgcgcccaa?gccgcgaggc 780

aggcaaagca?gggccgaggg?gagcccaagc?ccacacgccg?cacaccgggc?cccagagcca 840

cggagccacg?aacacccacc?acgagggcag?cggcggacgg?cagagccccg?ggccagccca 900

ccccggggac?agggggggac?cagcggggag?acacaagggc?gggaacacca?acccgggaac 960

aaaccgagag?agaaccccaa?cacacggagc?aagccccaga?aaagccaccc?cggacaaagg 1020

gcaaaccgaa?cgccgccaga?ggccacgcgc?cgccagccgc?ggagacaccc?cacccaaggc 1080

cccccacaga?ggccggcgca?cagccgagaa?cgcgagcggg?aacccagcgc?caacaaccgc 1140

ccaccccccc?ccaaccaggc?gggaacccca?ccaaccgccc?aacgccacca?ccccccacga 1200

ggccccagcg?ggcacaccac?cccaccccgc?ccacaagcaa?cgagacccga?ccagccagac 1260

ggcagcgacc?gagccacacc?accccacaac?ccccgagggc?cccaccaagc?acccccaccc 1320

acgggagccc?caagaggggc?gggaaggggg?gccaagccca?caagcccgcc?cggcgggccc 1380

cagacagagg?gcagaggaaa?gagcccgccc?caccccagcc?cccccaccag?cccaccccgg 1440

ggggcaagag?gaaacggggg?ggagggaaga?gaaggacagg?ggggggggag?aggaccccac 1500

ccccggcccc?cccccccccc?agaccccacc?ccccagaccc?ccccccccgg?cccgaaaaga 1560

accagcccag?cccgcccccc?ccccggcccc?cggggaaaag?agaaacaaag?aaaacgcgac 1620

gcaccgccaa?ccggccccgc?cccccacagc?gaaccccccc?gcccgccccc?aaggcacccc 1680

cccaccccac?ccggagggcc?aggggaggga?gagagcccga?gcagccacag?aagggccgga 1740

cagaccccgc?aaaaaaggca?gaaaaccgaa?aaaaaaaaaa?aaaaaaaaa 1789

 

<210>6

<211>433

<212>PRT

＜213〉people

 

<400>6

 

Met?Ser?Gly?Arg?Pro?Arg?Thr?Thr?Ser?Phe?Ala?Glu?Ser?Cys?Lys?Pro

1 5 10 15

Val?Gln?Gln?Pro?Ser?Ala?Phe?Gly?Ser?Met?Lys?Val?Ser?Arg?Asp?Lys

20 25 30

Asp?Gly?Ser?Lys?Val?Thr?Thr?Val?Val?Ala?Thr?Pro?Gly?Gln?Gly?Pro

35 40 45

Asp?Arg?Pro?Gln?Glu?Val?Ser?Tyr?Thr?Asp?Thr?Lys?Val?Ile?Gly?Asn

50 55 60

Gly?Ser?Phe?Gly?Val?Val?Tyr?Gln?Ala?Lys?Leu?Cys?Asp?Ser?Gly?Glu

65 70 75 80

Leu?Val?Ala?Ile?Lys?Lys?Val?Leu?Gln?Asp?Lys?Arg?Phe?Lys?Asn?Arg

85 90 95

Glu?Leu?Gln?Ile?Met?Arg?Lys?Leu?Asp?His?Cys?Asn?Ile?Val?Arg?Leu

100 105 110

Arg?Tyr?Phe?Phe?Tyr?Ser?Ser?Gly?Glu?Lys?Lys?Asp?Glu?Val?Tyr?Leu

115 120 125

Asn?Leu?Val?Leu?Asp?Tyr?Val?Pro?Glu?Thr?Val?Tyr?Arg?Val?Ala?Arg

130 135 140

His?Tyr?Ser?Arg?Ala?Lys?Gln?Thr?Leu?Pro?Val?Ile?Tyr?Val?Lys?Leu

145 150 155 160

Tyr?Met?Tyr?Gln?Leu?Phe?Arg?Ser?Leu?Ala?Tyr?Ile?His?Ser?Phe?Gly

165 170 175

Ile?Cys?His?Arg?Asp?Ile?Lys?Pro?Gln?Asn?Leu?Leu?Leu?Asp?Pro?Asp

180 185 190

Thr?Ala?Val?Leu?Lys?Leu?Cys?Asp?Phe?Gly?Ser?Ala?Lys?Gln?Leu?Val

195 200 205

Arg?Gly?Glu?Pro?Asn?Val?Ser?Tyr?Ile?Cys?Ser?Arg?Tyr?Tyr?Arg?Ala

210 215 220

Pro?Glu?Leu?Ile?Phe?Gly?Ala?Thr?Asp?Tyr?Thr?Ser?Ser?Ile?Asp?Val

225 230 235 240

Trp?Ser?Ala?Gly?Cys?Val?Leu?Ala?Glu?Leu?Leu?Leu?Gly?Gln?Pro?Ile

245 250 255

Phe?Pro?Gly?Asp?Ser?Gly?Val?Asp?Gln?Leu?Val?Glu?Ile?Ile?Lys?Val

260 265 270

Leu?Gly?Thr?Pro?Thr?Arg?Glu?Gln?Ile?Arg?Glu?Met?Asn?Pro?Asn?Tyr

275 280 285

Thr?Glu?Phe?Lys?Phe?Pro?Gln?Ile?Lys?Ala?His?Pro?Trp?Thr?Lys?Asp

290 295 300

Ser?Ser?Gly?Thr?Gly?His?Phe?Thr?Ser?Gly?Val?Arg?Val?Phe?Arg?Pro

305 310 315 320

Arg?Thr?Pro?Pro?Glu?Ala?Ile?Ala?Leu?Cys?Ser?Arg?Leu?Leu?Glu?Tyr

325 330 335

Thr?Pro?Thr?Ala?Arg?Leu?Thr?Pro?Leu?Glu?Ala?Cys?Ala?His?Ser?Phe

340 345 350

Phe?Asp?Glu?Leu?Arg?Asp?Pro?Asn?Val?Lys?Leu?Pro?Asn?Gly?Arg?Asp

355 360 365

Thr?Pro?Ala?Leu?Phe?Asn?Phe?Thr?Thr?Gln?Glu?Leu?Ser?Ser?Asn?Pro

370 375 380

Pro?Leu?Ala?Thr?Ile?Leu?Ile?Pro?Pro?His?Ala?Arg?Ile?Gln?Ala?Ala

385 390 395 400

Ala?Ser?Thr?Pro?Thr?Asn?Ala?Thr?Ala?Ala?Ser?Asp?Ala?Asn?Thr?Gly

405 410 415

Asp?Arg?Gly?Gln?Thr?Asn?Asn?Ala?Ala?Ser?Ala?Ser?Ala?Ser?Asn?Ser

420 425 430

Thr

 

<210>7

<211>2503

<212>RNA

＜213〉people

 

<400>7

gcggagcgcg?gcgcgcgcgg?cgggggccgg?gcgcggcccg?ggaccgccgc?cacccgcgac 60

agaccgccgg?ccgcgagccg?ccggagcacc?ggacaagccg?gcgccaacga?gccgggggca 120

cgcccgcagc?ggccaagcca?ggccggcgag?cgggacgccg?ccccgcccag?ccaccgccgc 180

cgccgccgcc?cccccccagc?cggcggcggc?ccgggcccag?caaccaggcg?aagacacggg 240

acgggcgccg?cggcgaacag?gaggagaagg?aggcgcgcgg?cccacccggg?ccgccgcccc 300

aggcgccgcc?gcgccggccc?cgcggccgag?ggccgcgcgc?ccccgccgac?gccaggacgg 360

agaagaagac?aaacggcagc?gcaagacacc?cgaggggccg?aggcaagaca?agaccaaggg 420

ccccgagcag?aaggccggag?agaggggggg?ggcggcagga?cccggagagg?cccccacacg 480

gcgcccgggg?aaccgcgcac?ggggcccacc?agccgcacca?gagaggcacg?aggacgaaga 540

ggggcgaggg?gagaggacca?ggccagccac?gcccggagag?ggcagcccag?gagaggcgag 600

cgcaaccacc?cccacgcaag?acccacgagg?acacaacaag?cgccacacac?cagcgacacc 660

ggcgccgagg?gcaccggaga?agcgacccca?aagccccacg?acaagccccc?agccgccgcc 720

ccgcggcagc?cacgagaggc?gggaaacgga?gaccacaaag?cggacaaacg?ggcgagggac 780

cagccaccgc?acaaaggcaa?aagcaagcca?cagacaaccg?ggcaccaagg?agacagacgg 840

aacagaagag?ggggcacccg?caccgccacc?gggaaggccc?gccaaggacc?caaacacgcc 900

aacacgacgc?acagacaacc?acacggagaa?gccccacccg?cgagaccgga?caaggaccga 960

agcagaccgg?agacggggaa?cacacaacag?cacaacggaa?acgccgccag?cgcccgggcc 1020

ggccacgcca?ccggcagaag?ggcacaccga?gacccaagcc?ccagaaccgc?cacaacgaga 1080

ggggagagcc?aagcggcgac?ggccggcccg?agccaagcaa?cccaacaaag?acaacccaag 1140

agggggacac?gggaccggcc?cccgacaccg?cgggccacgg?acacccacca?gagacagggg 1200

gggggcgcac?cagagaggcc?acaggccgcc?cccccgggcc?cacggggagg?aacagcacac 1260

cacccgacag?gaaccccaac?gaggagacgg?gccaggcacc?gccaacgagg?agcaagacaa 1320

caacacccca?agaccgagcc?gaggcccgag?ccacgcaccc?cgacgaagcg?acggggccga 1380

cccccaccaa?gcggcaggag?ggcgaaacgg?acccgcagag?gagccagaaa?caccacccca 1440

gcgggggagc?ggaccacaaa?cccgacacac?ccaagcacaa?aggagacagc?acaaaaggag 1500

gccagcccgg?ccgcgagccg?accaggcagg?ccagcccgcg?ggggacaccg?agcaagccac 1560

agaccgaggc?cccagcaggc?agcggcggag?ggagccacac?ccccacaggg?cagcccccaa 1620

cacaccccgc?acccgccacc?gccgagccag?caccgcccac?gccccgcgcc?gcccaaacac 1680

cccaccaggc?cgcaacccac?ccaggccgcg?cggggcaaca?aagcccacac?cccacggcgc 1740

gccgcggagg?ccggggacag?caggccggcc?agcccccaca?cgaggccagg?caccccccac 1800

aaccagcccc?caggaccaca?ccccacggcc?agccaggggc?cagagcagcc?caggcgggga 1860

ccgaccagac?aagagggaca?agccgagcga?ggcacccgcc?gcccgccgcc?ccaccgccca 1920

agggggccgg?cacagaaaaa?agagagaaag?caaaccgaac?agacggcggg?ccccaccccc 1980

gcggggcccc?ccacagaggc?aagaagcccg?ccccagccca?gagacaggga?cacagcccca 2040

ggaacccgac?acaccagacc?cgggaggcag?ggaaagcagc?cacagccacc?gccccacccc 2100

cgcccgccac?cccaggcagg?gggacgggga?caccagagac?cgggaaagga?caaggggggg 2160

gccccacccc?ccgcagcccg?agcggggccc?cccccagggc?ccccagccca?gccccgcccc 2220

acccaccggg?cggggagggg?cccgccagga?acgaccagcc?agcgaggagc?caaaggcaag 2280

gcacaagcag?gggggggagg?ggggggaggg?gggcccaggg?gcccccaccc?ggggagggga 2340

ggcagggcag?ggacagccca?gggcagcccg?gagggggacc?ccccccccac?ccaagcccgg 2400

ggcccgaaag?ggggggaggg?caggggggga?gcccccaggg?gggggggggg?ccgaagcacc 2460

aaacgcgaga?aaaaaaaagc?aaaggaaaaa?aaaaaaaaaa?aaa 2503

<210>8

<211>304

<212>RNA

＜213〉people

 

<220>

<221>misc_feature

<222>(16)..(16)

＜223〉n is a, c, g or u

 

<220>

<221>misc_feature

<222>(21)..(21)

＜223〉n is a, c, g or u

 

<220>

<221>misc_feature

<222>(24)..(24)

＜223〉n is a, c, g or u

 

<220>

<221>misc_feature

<222>(53)..(53)

＜223〉n is a, c, g or u

 

<220>

<221>misc_feature

<222>(60)..(60)

＜223〉n is a, c, g or u

 

<220>

<221>misc_feature

<222>(81)..(81)

＜223〉n is a, c, g or u

 

<220>

<221>misc_feature

<222>(89)..(89)

＜223〉n is a, c, g or u

 

<220>

<221>misc_feature

<222>(100)..(100)

＜223〉n is a, c, g or u

 

<220>

<221>misc_feature

<222>(131)..(131)

＜223〉n is a, c, g or u

 

<220>

<221>misc_feature

<222>(149)..(149)

＜223〉n is a, c, g or u

 

<220>

<221>misc_feature

<222>(170)..(170)

＜223〉n is a, c, g or u

 

<220>

<221>misc_feature

<222>(175)..(175)

＜223〉n is a, c, g or u

 

<220>

<221>misc_feature

<222>(193)..(194)

＜223〉n is a, c, g or u

 

<220>

<221>misc_feature

<222>(210)..(210)

＜223〉n is a, c, g or u

 

<220>

<221>misc_feature

<222>(247)..(247)

＜223〉n is a, c, g or u

 

<220>

<221>misc_feature

<222>(249)..(249)

＜223〉n is a, c, g or u

 

<220>

<221>misc_feature

<222>(251)..(251)

＜223〉n is a, c, g or u

 

<220>

<221>misc_feature

<222>(255)..(255)

＜223〉n is a, c, g or u

 

<220>

<221>misc_feature

<222>(292)..(292)

＜223〉n is a, c, g or u

 

<220>

<221>misc_feature

<222>(300)..(300)

＜223〉n is a, c, g or u

 

<400>8

mkkkrrsrgs?dssamnsskd?nvkngrshsm?hshygskkrr?hsvggsgsma?mrngsrdvhn 60

kmgsdgsdas?gssdvsgvcr?nrhrrsmdnk?rsadrdgykn?sdmsrrsrra?ssggkmykkg 120

gyavykgrsk?nvakrhgaca?rvskdkhanv?hdvhdksvyd?kdkymddcgn?msmhnvkyrg 180

aychrrkvhr?dknnkgkadg?araksvkysn?vvwyrdvgss?ysdmwgvgcm?asgrgsvdhr 240

gswgssnkny?nkyknhards?gkyskkrvsa?amkhvyrsgr?hasvsskkdg?rnssyghgkn 300

rrsm 304

 

<210>9

<211>1318

<212>RNA

＜213〉people

 

<400>9

ccccggcccg?agccaggcag?ccggcaggca?gaggcccacc?cgggccgggc?ccagaccggg 60

ggggcccgcc?aggaggggcc?gagcccgcac?gcccgaaaca?cgcagccgcg?cggcgcgagg 120

gcggggagcg?ggggcgccca?aagagggcgg?ggggaggaag?ggcccggagc?cgcgggccga 180

ggccgaaggc?gccgacccca?ccccgaagcg?gcgacggagc?agggccgcca?cacaagcaga 240

ccgcgggccc?ccagcgcacc?cggaagagcc?caccacccgg?acgagcccgg?agcccagacc 300

gccgaagagg?agacgagaca?agggccacgc?gcgggcgccc?ggggaacgca?cagacaaaag 360

cgcaccccac?cagaaacggg?ggaggggggc?agcagggacc?agggaaggga?cagaggacac 420

cacgcccgaa?gcgaaccggc?acgagcagca?ggaccgggag?gaggcccagc?gagaagccga 480

cagcacgccc?caacacccca?acacccgccc?gggcacgcga?gggaacgggg?gcaagcagag 540

gccggcgcgc?accaccaaga?gaggacgcgg?gaagagaaga?aaggcgaagg?acaaaggcaa 600

cccgaccgag?gacaaaccgg?cgcgcgggga?cgcagaggcc?gaggccacag?ccaagggagc 660

ccacagagac?gaagcccacc?aaaagcggag?agaggggcag?ccagaaggac?gggccagaac 720

aagcagcacc?agggagggcc?ccgccaggcc?gacccgcagg?acgggcagcc?cagcgggcac 780

cacccaccga?gccccagagc?cccggcagag?cacggcacga?gagcggacga?gcggcccagg 840

cgcggcaagc?cagaggggga?aggcccagac?agggccaaaa?ggggacaggg?gcccgcggca 900

gaaccaacca?gcacccacaa?agccccaggc?accagcagcg?gcagcccgaa?ccgagagacc 960

gggacccgca?cagcgcccac?acccccccca?gcagcggagg?cgcgcagccc?ccagcccggc 1020

caacaacacc?caaacgaaaa?agcagcagga?gaagaggccc?cggccggaaa?gaggcccacc 1080

ccaggaacac?cacccaccac?caggacccac?acgggggagc?ggggcaggac?aacaccagcc 1140

gcacccgccc?ccccaagagc?aaaaccgggc?aaggggacac?gagggggggg?ggggggggga 1200

aaagggaaac?gggggaagga?acaggccgag?caggacggag?ccacaaggcg?acccaaacgg 1260

gagcaggaga?aggaaacaag?aaaaagggaa?gcagggggag?aaaaaaaaaa?aaaaaaaa 1318

Claims (12)

1. the method for a predictive compound genotoxicity, described method comprises:

A) provide test compounds;

B) measure the ability that this compound suppresses at least 10 kinds of kinase whose kinase activities, described kinases is selected from the group of being made up of CDK2, CLK1, DYRK1B, ERK8, GSK3A, GSK3B, PCTK1, PCTK2, STK16, TTK, CLK2, ERK3 and PRKR, and wherein at least 5 kinds of described kinases are suppressed at least 50% activity and show that described test compounds has genotoxicity.

2. the method for a predictive compound genotoxicity, described method comprises:

A) provide test compounds;

B) measure the ability that this compound suppresses at least 10 kinds of kinase whose kinase activities, described kinases is selected from the group of being made up of CDK2, CLK1, DYRK1B, ERK8 (MAPK15), GSK3A, GSK3B, PCTK1, PCTK2, STK16, TTK, CDK7, CLK4 and PCTK3, and wherein at least 5 kinds of described kinases are suppressed at least 50% activity and show that described test compounds has genotoxicity.

3. method according to claim 1 and 2, wherein step b) further comprises the ability of measuring at least a kind of kinase whose kinase activity of this compound inhibition, and described kinases is selected from by MKNK2, SgK085, PIM2, TNNI3K, KIT, MELK, AURKA, CLK3, AAK1, DCAMKL3, LIMK1, FLT1, MAP2K4, PIM3, AURKB, ERK2, CSNK1A1L, DAPK3, MLCK, CLK3, PFTK1, PRKD3, AURKC, ERK5, STK17A, MST4, CDK3, MYLK, CDC2L1, QIK, CDK11, PLK1, PDGFR β, PRKCM, MAPK4, PIP5K2B, CSNK1D, RPS6KA1.Kin.Dom.1, CDK5, PLK3, BIKE, PLK4, CAMK2A, STK3, CSNK2A1, STK17B, CDK8, MAP2K6, PIM1, MAP2K3, CDK7, IKK ε, TGFBR2, CDK9, the group that CLK4 and PCTK3 form.

4. according to the described method of claim 1 to 3, wherein said test compounds is tested in the concentration of about 10 μ M.

5. according to the described method of claim 1 to 4, wherein step b) comprises the ability that this compound inhibition is selected from least 12 kinds of kinase whose kinase activities of described group of measuring.

6. according to the described method of claim 1 to 4, wherein step b) comprises the ability that this compound suppresses whole 13 kinds of kinase whose kinase activities in described group of measuring.

7. screen the method for latent gene toxic chemical, described method comprises:

A) provide the substantive test compound;

B) measure the ability that each compound suppresses at least 10 kinds of kinase whose kinase activities, described kinases is selected from the group of being made up of CDK2, CLK1, DYRK1B, ERK8, GSK3A, GSK3B, PCTK1, PCTK2, STK16, TTK, CLK2, ERK3 and PRKR, or is selected from other groups of being made up of CDK2, CLK1, DYRK1B, ERK8 (MAPK15), GSK3A, GSK3B, PCTK1, PCTK2, STK16, TTK, CDK7, CLK4 and PCTK3;

Wherein at least 5 kinds of described kinases are suppressed at least 50% activity and show that described test compounds has genotoxicity.

8. method according to claim 7 further comprises:

C) the refusal proof may have the compound of genotoxicity.

9. according to the described method of claim 1 to 8, wherein this compound ability of suppressing kinase activity is by measuring this compound and described kinase whose binding affinity is measured.

10. test substrate for one kind, it comprises:

Solid support; And

Be fixed on kinase c DK2, CLK1, DYRK1B, ERK8, GSK3A, GSK3B, PCTK1, PCTK2, STK16, TTK, CLK2, ERK3 and PRKR on the described solid support, or kinase c DK2, CLK1, DYRK1B, ERK8 (MAPK15), GSK3A, GSK3B, PCTK1, PCTK2, STK16, TTK, CDK7, CLK4 and PCTK3.

11. test substrate according to claim 10 further comprises:

Be fixed on the kinases on the described solid support, described kinases is selected from by MKNK2, SgK085, PIM2, TNNI3K, KIT, MELK, AURKA, CLK3, AAK1, DCAMKL3, LIMK1, FLT1, MAP2K4, PIM3, AURKB, ERK2, CSNK1A1L, DAPK3, MLCK, CLK3, PFTK1, PRKD3, AURKC, ERK5, STK17A, MST4, CDK3, MYLK, CDC2L1, QIK, CDK11, PLK1, PDGFR β, PRKCM, MAPK4, PIP5K2B, CSNK1D, RPS6KA1.KD1, CDK5, PLK3, BIKE, PLK4, CAMK2A, STK3, CSNK2A1, STK17B, CDK8, MAP2K6, PIM1, MAP2K3, CDK7, IKK ε, TGFBR2, CDK9, the group that CLK4 and PCTK3 form.

12. in fact as mentioned above, particularly with reference to the method for previous embodiment and test substrate.