CN101052646A - Cell cycle phase markers - Google Patents

Abstract

The present invention relates to nucleic acid reporter constructs and host cells transfected with said constructs. The invention also relates to methods which are useful for determining the cell cycle status of a mammalian cell and for determining the effect of a test agent on the cell cycle position of a mammalian cell.

Description

Cell cycle phase markers

Technical field

The method that the present invention relates to cell cycle phase specificity marker thing and be used for determining the transformation between the mammalian cell cell cycle different steps.

Background of invention

The eukaryotic cell division was undertaken by the cell cycle that is called the successive stage of G1, S, G2 and M comprising of high degree of controlled.Cell cycle or the cell cycle destruction of control can cause cellular abnormality or morbid state such as cancer, and this results from multiple hereditary change, and its cell transformation with growth restriction has become highly invasive cell, and they are not replied the normal control of growth.Normal cell to the transformation of cancer cells can be by dna replication dna and DNA repair mechanism the forfeiture of correct function produce.Cell in all divisions all is controlled by many controlling mechanisms, is called cell cycle chechpoint, and it is complete by stagnating abnormal cells or inducing paracytic destruction to keep genome.Therefore the research of cell cycle progress and control is (Flatt, P.M. and Pietenpol, J.A.Drug Metab.Rev., (2000), 32 (3-4), the 283-305 that suitable importance is arranged for the design cancer therapy drug; Buolamwini, J.K.Current Pharmaceutical Design, (2000), 6,379-392).

The cell cycle progress is by the specified time of many Cycle Regulation agent and space expression, location and destroys strict control that they present dynamic (Pines, the J. of height during the cell cycle, Nature Cell Biology, (1999), 1, E73-E79).For example, at specific cell cycle phase, some protein translocate to tenuigenin from nucleus, and are perhaps opposite, and some protein are degraded rapidly.Understand known cell cycle control composition and interactional details, referring to Kohn, Molecular Biology of the Cell (1999), 10,2703-2734.

The research of accurately determining of cell cycle state influences the key request of the cell processes of cell cycle or the cell cycle of depending on position.These are determined in the drug screening application is vital especially, wherein:

I) need the direct or indirect material that changes the cell cycle progress, for example be used for research as the potential anticancer therapy;

Ii) check the harmful effect of drug candidate cell cycle progress; And/or

Suspect that iii) reagent is active or non-activity to the cell in the specific cells phase of the cycles.

Traditionally, the cell cycle state of cell mass determines by flow cytometry, wherein make the nuclear DNA of fluorochromine (Barlogie, B.et al, Cancer Res., (1983), 43 (9), 3982-97).Flow cytometry produces the quantitative information of cell DNA content, therefore can determine to be in G1, the S of cell cycle and the relative cell quantity of G2+M phase.Yet this analysis is destructive non-dynamic process, needs the serial sampling of cell mass to determine time dependent cell cycle state.More shortcomings of Flow Cytometry relate to cell cycle position indirect according to dna content and definite cell inferential.Because nuclear dna content changed in quite expected mode in the whole cell cycle, promptly be in the dna content that the cell of G2 or M has G1 cell twice, being in the S phase carries out the DNA that DNA synthetic cell has intermediate quantity, and it is possible therefore monitoring the relative distribution of cell between the cell cycle different steps.Yet, this technology can not accurately be determined the cell cycle position of any individual cells, this be since with cell belong to the uncertain of G2 or M phase and since in the cell mass between cell and the cell dna content that intrinsic difference caused was further inaccurate, this may get rid of the accurate differentiation between the cell that the boundary between the adjacent phase with the cell cycle is close.In addition, dna content and this technology of the painted difference requirements of DNA between the different cell types of different tissues or biology are optimized every kind of cell type, may make between the cell type or the immediate data more complicatedization (Herman between the experiment, Cancer (1992), 69 (6), 1553-1556).Therefore flow cytometry is suitable for checking the whole cell period profile of cell in the cell mass, but can not be used to monitor the time dependent accurate cell cycle state of individual cells.

EP 798386 has described the method for the cell cycle that is used for analyzing the cell subsets that the foreign cell sample exists.What this method was used is to differentiate the particular cell types and the fluorescence dye of specificity bind nucleic acid with the continuous incubation sample of fluorescently-labeled monoclonal antibody.This makes it possible to determine the cell cycle distribution of the cell subsets that exists in the sample.Yet, because this method has been utilized flow cytometry, therefore it only produces non-dynamic data, after the reagent that need study being exposed to, on independent cell sample, carry out cell cycle state that METHOD FOR CONTINUOUS DETERMINATION determines cell mass over time, to determine the influence of cell cycle progress.

Many investigators have used and cell fixation or molten born of the same parents' routine report enzyme need have been studied the cell cycle.For example, Hauser﹠amp; Bauer (Plant and Soil, (2000), 226,1-10) used β-glucuronidase (GUS) to study cell fission in the plant meristematic tissue, Brandeis﹠amp; Hunt (EMBO J., (1996), 15,5280-5289) used E.C. 2.3.1.28 (CAT) fusion rotein to study the variation of cyclin white level.US 6048693 has described the method that is used to screen the compound that influences cyclin, and wherein the expression with reporter gene is connected with controlling elements, and these elements work by cyclin or other cell cycle control albumen.In the method, it is driven in special mode of cell cycle that the timeliness of reporter gene product is expressed, and the compound that acts on one or more cell cycle control compositions can increase or reduce expression level.

US 6159691 has described the nuclear localization signal (NLS) that is derived from cell cycle phase idiosyncratic transcription factor DP-3 and E2F-1, the claimed method of inferring conditioning agent that is used for the analysis of cells cycle progress.In the method, the nuclear localization signal (NLS) that is derived from cell cycle phase idiosyncratic transcription factor DP-3 and E2F-1 can be used for the analysis of compounds activity, and this compound is used for increasing or reduce the position of appraising and deciding from the specific NLS sequence of DP-3 and E2F-1 that is fused on the detectable label.

Jones et al (Nat Biotech., (2004), 23,306-312) mitotic biological sensor has been described, it is based on plasma membrane target signal and the SV40 large T antigen NLS that is fused on the EYFP.In the whole cell cycle, reporter gene all is arranged in nucleus, but during the mitotic division, nuclear membrane decomposes and form again between translocated on the plasma membrane.

WO 03/031612 has described DNA report construct and the method for the cell cycle position of the mammalian cell that is used for determining living, and it relies on the specific expressed controlling elements of cell cycle phase and destroys controlling elements.

Gu et al. (Mol Biol Cell., 2004,15,3320-3332) recently after deliberation the function of people's dna helicase B (HDHB), show it the G1 phase substantially at nucleus, S and G2 phase substantially at tenuigenin, dna damage induces it to be positioned at epipole, this epipole pattern needs the HDHB activity, and the HDHB location is controlled by the CDK phosphorylation.

Aforesaid method is not all clearly described to be stabilized and is incorporated into G1, S and the transmitter of G2 phase that is used to indicate the cell cycle in the genome.Therefore, need a kind of method, it makes these cell cycle phases to be determined by nondestructive ground in single mammalian cell alive, make that same cell can be repeated to detect in time, and make it possible to study that cell cycle has the potential expectation or the influence of the reagent not expecting to influence.Also need to carry out these influences of plurality of reagents the method for parallel evaluation.

Summary of the invention

The invention describes a kind of method, it utilizes the key component of cell cycle controlling mechanism that new means are provided with the combination of regulation, determines the cell cycle state of individual viable cell with the nondestructive process that dynamic reading is provided.

The present invention further provides protein, DNA construct, carrier and express these proteinic stable cell lines, they present the transposition that can detect reporter molecule in cell cycle phase specificity mode, and it is directly connected to G1/S cell cycle phase dependency setting control sequence by reporting signal.This has greatly improved the accuracy of definite cell cycle phase state, and makes it possible to face continuously the cell cycle progress of surveying in the individual cells.And, have been found that and to separate and extract crucial controlling elements by the functional element of cell cycle controlling mechanism, this makes it possible to design dynamically controlled cell cycle phase reporter molecule, but and can as one man operate independently with endogenous cell periodic Control composition, thereby the means that are used to monitor the cell cycle state are provided, its do not influence or the interference cell cycle naturally the progress.

According to a first aspect of the invention, polypeptide construct is provided, it comprises detectable viable cell reporter molecule, this molecule is by having less than 112, the group of 000 Dalton molecular weight is connected at least one cell cycle phase dependency positioning controling element, being positioned at G1 and changing during the S phase of described element, the transposition of wherein said construct in mammalian cell shows the cell cycle position.

It being understood that transposition is defined as reporter molecule and can moves to another from a Subcellular Localization with detecting, typically from the nucleus to tenuigenin or opposite.To further be understood that, when relating to reporter molecule, term " viable cell " has defined the reporter molecule that produces detectable signal in viable cell, perhaps such as the such reporter molecule of antigenicity sign, it is expressed in viable cell and can be detected after fixing by immunological method, so just be suitable in the imaging system, such as IN Cell Analyzer (GE Healthcare).

Suitably, described group has less than 100,000 daltonian molecular weight.

Suitably, group has less than 50,000 daltonian molecular weight.

Suitably, group has less than 25,000 daltonian molecular weight.

Suitably, group has less than 10,000 daltonian molecular weight.

Suitably, group has less than 1,000 daltonian molecular weight.

Suitably, group has less than 700 daltonian molecular weight.

Suitably, group has less than 500 daltonian molecular weight.

Preferably, group is a polypeptide.Peptide group should be relatively little, and comprise the amino acid that allows reporter molecule to have flexibility and/or can rotate with respect to cell cycle phase dependency positioning controling element.More preferably, peptide group is seven peptides.Most preferably, described seven peptide groups are glycine-l-asparagine-glycine-glycine-l-asparagine-L-Ala-Serine (GNGGNAS).As mentioned above, in polypeptide, can use any amino acid that allows reporter molecule to have flexibility and/or can rotate with respect to positioning controling element.

Suitably, cell cycle phase specific localization controlling elements is selected from the peptide group of being made up of Rag2, Chaf1B, Fen1, PPP1 R2, helicase B, sgk, CDC6 or its motif, the phosphorylation dependency Subcellular Localization structural domain (PSLD) in the C-terminal spatial control district of described motif such as helicase B.Known helicase B causes not controlled DNA permission and may the pair cell survival be harmful to when overexpression.Therefore, preferably, cell cycle phase dependency positioning controling element is the phosphorylation dependency Subcellular Localization structural domain (PSLD) in the C-terminal spatial control district of helicase B.

Reported and characterized people's helicase B homologue (Taneja et al J.Biol.Chem., (2002), 277,40853-40861); Nucleotide sequence (NM 033647) and corresponding proteins matter sequence are shown in SEQ ID No.1 and SEQ ID No.2 respectively.The report proof needs helicase activity to promote G1/S to change during G1.Gu et al (Mol.Biol.Cell., (2004), 15,3320-3332) show, NLS and NES sequence are contained by Cdk2/ cyclin E phosphorylation in the little C-terminal district of helicase B gene that is called phosphorylation dependency Subcellular Localization structural domain (PSLD).Gu et al (Mol.Biol.Cell., (2004), 15,3320-3332) on the cell of using by the plasmid transient transfection of the coding EGFP-β Gal-PSLD fusions of CMV promoter expression (in construct, comprise beta-galactosidase enzymes (β Gal) as inertia group so that whole fusion rotein is similar to helicase B in size), study.The cell that is in G1 mainly shows the EGFP signal in nucleus, and is in the main showed cell matter of the cell EGFP signal of other cell cycle phase.These investigators conclude, before and after the G1/S phase of cell cycle changes, PSLD guided reporter molecule from nucleus to cytoplasmic transposition.

Suitably, the viable cell reporter molecule is selected from and comprises fluorescin, enzyme and antigenicity sign.Preferably, fluorescin be derived from Aequoria Victoria, Renilla reniformis or Hydrozoa and Anthozoa other member (Labas et al., Proc.Natl.Acad.Sci, (2002), 99,4256-4261).More preferably, fluorescin be EGFP (BD Clontech), Emerald (Tsien, Annu.Revs.Biochem., (1998), 67,509-544) or J-Red (Evrogen).Most preferably, fluorescin is selected from green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Emerald and J-Red.

Preferably, reporter molecule is the enzyme reporter molecule such as halo-tag (Promega).

Suitably, reporter molecule is EGFP or J-Red, and cell cycle phase dependency positioning controling element is PSLD.

Suitably, reporter molecule is placed in-line (promptly being rendered as series connection repeats).

Polypeptide construct comprises the aminoacid sequence of SEQ ID No.5.

According to a second aspect of the invention, provide the coding nucleic acid construct of any polypeptide construct as mentioned above.

Suitably, described nucleic acid construct additionally comprises and is operably connected to and is controlled by at least one dependent/non-dependent cell cycle and expresses controlling elements.

Term " be operably connected " expression element arrangement make they and its intended purposes as one man work, for example initial transcribing in promotor carried out the dna sequence dna by code book invention reporter molecule.

Suitably, the expression controlling elements is controlled in the secular time period and is transcribed, and the transcriptional level of whole cell cycle is limited variable.Preferably, expressing controlling elements is ubiquitin C or CMVI/E promotor, and it provides, and stable cell lines production is needed transcribes for a long time.

Preferably, nucleic acid construct comprises the sequence of ubiquitin C promotor and coding PSLD and EGFP or J-Red.

Randomly, nucleic acid construct comprises the sequence of CMV promotor and coding PSLD and EGFP or J-Red.

In a third aspect of the present invention, provide to comprise as mentioned above the carrier of nucleic acid construct arbitrarily.Suitably, described carrier is virus vector or plasmid.Suitably, described virus vector is adenovirus carrier or lentiviral vectors.

Randomly, the carrier extra packet is contained in the drug resistance gene that function is arranged in the eukaryotic cell, and the drug resistance gene of function is preferably arranged in mammalian cell.

Expression vector also can comprise other nucleotide sequence, such as poly-adenosine signal, donor splicing site/acceptor splicing site signal, intervening sequence, transcriptional enhancer sequence, translational enhancer sequence or the like.Randomly, drug resistance gene and reporter gene can pass through internal ribosome entry site (IRES) (Janget al., J.Virology, (1988), 62,2636-2643) be operably connected, rather than by two genes of the promoters driven of separating.Can use commercial pIRES-neo and the pIRES carrier that obtains from Clontech.

A fourth aspect of the present invention provides the host cell with aforesaid nucleic acid construct transfection.The host cell of having introduced this construct therein or having contained the expression vector of this construct can be any mammalian cell that can express this construct.

Can use the technology of well known to a person skilled in the art with the DNA transfection of reporter constructs for preparing in host cell.These technology can comprise: electroporation (Tur-Kaspa et al, Mol.Cell Biol. (1986), 6,716-718), based on the method for calcium phosphate (for example Graham and Vander Eb, Virology, (1973), 52,456-467), directly microinjection, method (transgenosis (the Jiao et al that for example uses Superfect (Qiagen) or Fugene6 (Roche) and use the bombardment mediation based on cation lipid, Biotechnology, (1993) 11, 497-502).Be used for the DNA construct transfection is entered cell to the natural ability of more alternative approach utilization viruses of cell.Carrier that these methods comprise and transfection scheme are based on for example hsv (United States Patent (USP) 5288641), cytomegalovirus (Miller, Curr.Top.Microbiol.Immunol., (1992), 158,1), vaccinia virus (Baichwal and Sugden, 1986, in Gene Transfer, ed.R.Kucherlapati, New York, Plenum Press, p117-148) and adenovirus and adeno associated virus (Muzyczka, Curr.Top.Microbiol.Immunol., (1992), 158,97-129).

The example of suitable recombinant host cell comprises the mammal cell line of HeLa cell, Vera cell, Chinese hamster ovary cell (CHO), U2OS, COS, BHK, HepG2, NIH 3T3 MDCK, RIN, HEK293 and other vitro culture.Preferred host cell is people's cell.These clones can be available from American type culture collection (ATCC), Bethesda, Maryland, U.S.A.The present invention expects also and comprises cell from primary cell line that then limited for some time of culturing cell is established these clones after Mammals takes off cell.

In preferred embodiments, clone is the stable cell lines that comprises according to the multiple host cell of fourth aspect.

The clone that also can use standard method will show the stably express of cell cycle position reporter molecule is used for setting up the heterograft (Krasagakis of through engineering approaches cell in host animal, KJ et al, Cell Physiol., (2001), 187 (3), 386-91; Paris, S.et al, Clin.Exp.Metastasis, (1999), 17 (10), 817-22).Through engineering approaches will make it possible to set up model system with the tumor cell line heterograft of express cell period position reporter molecule and study tumour cell division, stagnation and transfer and screen new cancer therapy drug.

In a fifth aspect of the present invention, the purposes of aforesaid polypeptide is provided, be used for determining the cell cycle position of mammalian cell.

As the allograft in the host animal, this can study the tolerance that influences tissue transplantation or the mechanism (Pye﹠amp of repulsion with the through engineering approaches clone of express cell period position reporter molecule or genetically modified organism; Watt, J.Anat., (2001), 198 (Pt 2), 163-73; Brod, S.A.et al, Transplantation (2000), 69 (10), 2162-6).

According to a sixth aspect of the invention, provide the method for the cell cycle position that is used for definite mammalian cell, described method comprises:

A) in cell, express aforesaid nucleic acid construct; And

B) signal of launching by the monitoring reporter molecule is determined the cell cycle position.

For carrying out the method for cell cycle position that is used for determining cell according to the 6th aspect, can be with transfection the cell of DNA reporter constructs cultivate for some time under certain condition, it is enough to make the specified phase in the cell cycle to express reporter molecule.Typically, the expression of reporter molecule will occur in after the transfection 16 to 72 hours, but may change according to culture condition.If reporter molecule is based on the green fluorescent protein sequence, the time that reporter molecule may need to determine is folded into the conformation of fluorescence.This time is depended on the primary sequence of employed green fluorescent protein derivative.Fluorescence report albumen also can change in time color (referring to for example Terskikh, Science, (2000), 290,1585-8), this need be with the particular time interval imaging after transfection.

If reporter molecule has produced the fluorescent signal in the method for the 6th aspect, just can use conventional fluorescent microscope or monitor the signal of emission based on confocal fluorescent microscope.Use these technology, just can determine to express the cell proportion of reporter molecule, and the location of reporter molecule.In the method according to the invention, can be suitably by optical instrument in cell sorter, confocal microscope or the scanning confocal equipment of for example spectrophotometer, photofluorometer, fluorescent microscope, cooled charge-coupled device (CCD) imager (such as scanning imaging instrument or area imaging instrument), fluorescence excitation, measure transformed or transfection the fluorescence of cell of DNA construct, wherein can excite and launch the spectral quality of determining cell in the substratum by scan light.

In embodiments of the invention, nucleic acid reporter constructs wherein comprises drug resistance gene, in transfection with after expressing drug resistance gene (1-2 days usually), can select to express the cell of the reporter gene of modified by cell is grown in the presence of microbiotic, because the existence of selection marker thing gene makes cells transfected have resistance to antibiosis.Add antibiotic purpose and be the cell of selecting to express reporter gene, these cells in some cases with reporter gene together with it associating promotor be incorporated in the genome of clone.After the selection, can use standard technique to come the cloned cell line of separating table expression constructs.Cloned cell line can be grown under standard conditions then, and will express reporter molecule and produce detectable signal at the specified point of cell cycle.

Can cultivate transfection under the situation of test agent to be studied according to the cell of nucleic acid reporter constructs of the present invention lacking and/or exist, the influence of the cell cycle of described reagent pair cell is to be determined.The ratio of the cell by determine expressing reporter molecule and the location of the signal in the cell, the influence of determining the cell cycle of test agent pair cell is possible, for example whether test macro stagnates cell in the specified phase of cell cycle, and perhaps influence is to quicken or the deceleration cell fission.

Therefore, according to a seventh aspect of the invention, provide the method for definite test agent to the influence of the cell cycle position of mammalian cell, this method comprises:

A) express aforesaid nucleic acid reporter constructs under the situation of test agent lacking and exist; And

B) determine the cell cycle position by the signal launched of monitoring reporter molecule, wherein the difference between measured the transmitting has been indicated the influence of the cell cycle position of test agent pair cell when lacking and have test agent.

Term " test agent " should be understood that the form or the chemical entities of electromagnetic radiation.Preferably, test agent is the chemical entities that is selected from medicine, nucleic acid, hormone, protein and peptide.Test agent can be that exogenous application arrives cell, perhaps can be peptide or protein expressed in the cell of studying.

In a eighth aspect of the present invention, the method for definite test agent to the influence of the cell cycle position of mammalian cell is provided, this method comprises:

A) in described cell, express aforesaid nucleic acid reporter constructs existing under the situation of described test agent;

B) signal of launching by the monitoring reporter molecule is determined the cell cycle position, and

The given value of the signal that the signal of being launched when c) relatively having test agent is launched when lacking test agent;

Wherein when having test agent measured transmit and the given value when lacking test agent between difference indicated the influence of the cell cycle position of test agent pair cell.

In a ninth aspect of the present invention, the method for definite test agent to the influence of the cell cycle state of mammalian cell is provided, this method comprises:

A) provide and contain the cell of nucleic acid reporter constructs as mentioned above;

B) under the condition that has and lack test agent and permission express nucleic acid reporter constructs, cultivate first and second cell masses respectively; And

C) measure the signal that the reporter molecule in first and second cell masses is launched;

Wherein the difference between measured the transmitting has been indicated the influence of the cell cycle position of test agent pair cell in first and second cell masses.

According to the tenth aspect of the invention, provide the method for the influence of definite mammalian cell cycle pair cell process, described process can first can detect reporter molecule and measures in what test agent changed by known response, and this method comprises:

A) in cell, express the aforesaid second nucleic acid reporter constructs existing under the situation of test agent;

B) determine the cell cycle position by monitoring the signal that second reporter molecule launches; And

C) monitoring first can detect the signal that reporter molecule is launched,

Wherein the determined cell cycle position of step b) and first relation that can detect between the signal that reporter molecule launches has indicated whether described cell processes is the cell cycle dependency.

In a eleventh aspect of the present invention, provide aforesaid polypeptide to be used for measuring the active purposes of CDK2 of cell.

According to a twelfth aspect of the invention, provide the active method of CDK2 in the mensuration cell, said method comprising the steps of

A) express nucleic acid construct in aforesaid cell, and

B) signal of launching by the monitoring reporter molecule is determined the CDK2 activity.

According to a thirteenth aspect of the invention, provide to be used for determining the method for test agent to the active influence of CDK2 of mammalian cell, described method comprises:

A) in described cell, express aforesaid nucleic acid construct under the situation of described test agent lacking and exist; And

B) determine the CDK2 activity by the signal launched of monitoring reporter molecule, wherein the difference between measured the transmitting has indicated test agent to the active influence of CDK2 in shortage and when having described test agent.

In a fourteenth aspect of the present invention, the method for definite test agent to the active influence of mammalian cell CDK2 is provided, described method comprises:

A) in described cell, express aforesaid nucleic acid construct existing under the situation of described test agent; And

B) determine the cell cycle position by the signal of monitoring reporter molecule emission,

The given value that the signal of being launched when c) relatively having test agent is transmitted when lacking test agent;

When wherein having test agent measured transmit and the described given value when lacking test agent between difference indicated the active influence of test agent pair cell CDK2.

The accompanying drawing summary

Further set forth the present invention with reference to the following examples and accompanying drawing, wherein:

The location of Fig. 1-HDHB in nucleus or tenuigenin.

(A) analyze the tenuigenin and the nucleus extract of U2OS cell, it is by denaturing gel electrophoresis and the western trace that carries out with anti-antibody of recombinating HDHB, alpha-tubulin and PCNA.By the chemiluminescence detection immunoreactive protein.

(B) observe microinjection in the U2OS cell and the HDHB of the GFP mark of transient expression by fluorescent microscopy.With Hoechst dyeing nucleus.Proportional sizes, 10 μ m.

(C) observe microinjection in the U2OS cell and the HDHB of the FLAG mark of transient expression by fluorescent microscopy.

The Subcellular Localization of Fig. 2-GFP-HDHB is the cell cycle dependency.

(A) Subcellular Localization of the HDHB of the GFP mark of transient expression in quantitatively asynchronous, G1 and the S phase U2OS cell.The quantity that will have the GFP positive cell of specific distribution pattern is expressed as the per-cent of GFP positive cell sum (＞100 cells).

(B) analyze synchronous U2OS cell (G1 and S phase) tenuigenin and nucleus extract, it is by denaturing gel electrophoresis and the western trace that carries out with anti-antibody of recombinating HDHB, alpha-tubulin and PCNA.By the chemiluminescence detection immunoreactive protein.

The affirmation in Fig. 3-desired structure territory, HDHB nucleus location.

(A) the proteinic synoptic diagram of HDHB has shown seven potential CDK phosphorylation sites (SP or TP), the Subcellular Localization structural domain (SLD) of inferring and phosphorylation SLD (PSLD), walker A and walker B helicase motif.Shown total number of atnino acid below the protein.

(B) GFP that produces in the research and the HDHB and the C-terminal truncated mutant of FLAG mark.The C-terminal of HDHB SLD (residue 1040-1087) and PSLD (residue 957-1087) is fused on the GFP-β Gal reporter molecule to form GFP-β Gal-SLD and GFP-β Gal-PSLD respectively.

(C) Subcellular Localization of the GFP-HDHB-Δ SLD of transient expression in quantitatively asynchronous, G1 and the S phase U2OS cell, and be expressed as the per-cent of GFP positive cell sum.

Fig. 4-GFP-β Gal-PSLD Subcellular Localization pattern is with the variation of cell cycle.

(A) GFP-β Gal, the GFP-β Gal-SLD of transient expression and the Subcellular Localization of GFP-β Gal-PSLD in quantitatively asynchronous, G1 and the S phase U2OS cell, and be expressed as the per-cent of GFP positive cell sum.

The discriminating of the functional rev type nuclear export signal (NES) among the SLD of Fig. 5-HDHB.

(A) infer NES (Henderson and Eleftheriou, 2000 of identifying in NES and other cell cycle related protein among the HDHB; Fabbro and Henderson, 2003) comparison.The subscript on aminoacid sequence top is represented the residue number.Conservative aliphatic residue among the thick arrow points NES.Two pairs of residues of inferring among the HDHB among the NES are sported L-Ala, shown in thin arrow, form Mut1 and Mut2.

(B) with (+) or need not (-) LMB in the U2OS of asynchronous growth cell the HDHB of transient expression GFP and FLAG mark to suppress the nuclear output of CRM1 mediation.The Subcellular Localization of GFP-HDHB and FLAG-HDHB in quantitatively asynchronous, G1 and the S phase cell, and be expressed as the per-cent of GFP positive cell sum in this sample.

(C) GFP-HDHB of wild-type and sudden change and the Subcellular Localization of GFP-β Gal-PSLD in quantitative nonsynchronous USOS cell, and be expressed as the per-cent of GFP positive cell sum in this sample.

Fig. 6-cell cycle relies on FLAG-HDHB phosphorylation in the gonosome.

(A) use [ ³²P] the U2OS cell of orthophosphoric acid salt mark transient expression FLAG-HDHB (swimming lane 1) and truncated mutant 1-1039 (swimming lane 2) and 1-874 (swimming lane 3).With anti-FLAG resin immunoprecipitation cell extract.Through the protein of 7.5%SDS-PAGE precipitation separation, shift on the road pvdf membrane, by radioautograph (on) or western trace (descend) detection.The position indication on the left of the marker protein matter of known molecular amount.

(B) with the FLAG-HDHB that expresses in the anti-FLAG resin immunoprecipitation U2OS cell, have (+) or lack under the situation of (-) inhibitors of phosphatases with (+) or need not (-) λ-Phosphoric acid esterase (incubation of λ-PPase), such as indicated, analyze and with anti-HDHB antibody mediated immunity trace through SDS-PAGE.

(C) the U2OS cell that will express FLAG-HDHB be stuck in G1/S (on) or G2/M (descending), remove blocking-up then.At the time point results FLAG-HDHB of indication,,, as in (B), analyze with (+) or need not (-) λ-PPase processing with anti-FLAG resin immunoprecipitation.

Fig. 7-affirmation S967 is the interior phosphorylation site of body main among the HDHB.

(A) phosphoamino acid (right side) of the FLAG-HDHB of two dimensional separation phosphoamino acid mark (left side) and ex vivo 32P mark passes through autoradiography observation.The phospho-peptide of some incomplete hydrolysis is retained near the original place (+).

(B) with radio-labeled wild-type in the orthophosphoric acid salt body and mutant FLAG-HDHB protein, immunoprecipitation separates through SDS-PAGE, by radioautograph (on) and analyze with anti-HDHB immunoblotting (descending).

(C) phospho-peptide of the trypsin hydrolyzing of the wild-type of two dimensional separation 32P mark and S967A mutant FLAG-HDHB passes through autoradiography observation.

Fig. 8-affirmation cyclin E/CDK2 is a HDHB S967 potential G1/S kinases.

(A) two dimensional separation is come among Fig. 7 C freely the phospho-peptide of the trypsin hydrolyzing of the FLAG-HDHB of phosphorylation in the body or purified cyclin E/CDK2 or the external phosphorylation reorganization of cyclin A/CDK2 HDHB, separate separately or, pass through autoradiography observation as mixture.

(B) by analyze that express in the U2OS cell and protein FLAG carrier (swimming lane 1,4) or FLAG-HDHB (swimming lane 2,5) co-immunoprecipitation with the antibody mediated immunity trace of anti-HDHB (swimming lane 1-6), cyclin E (swimming lane 1-3) or cyclin A (swimming lane 4-6).To be used for immunoprecipitation the molten born of the same parents' thing of cell 1/10th carry out parallel analysis (swimming lane 3,6) as positive control.

The Subcellular Localization of Fig. 9-HDHB is subjected to the control of S967 phosphorylation.

(A) GFP-HDHBS967A that expresses in quantitatively asynchronous, G1 and the S phase U2OS cell and the Subcellular Localization of S967D.

The location of EGFP-PSLD is the cell cycle dependency in the asynchronous U2OS cell of the stably express of Figure 10-demonstration pCORON1002-EGFP-C1-PSLD carrier.Fluorescent microscopy is carried out in same a part of visual field of pair cell, and wherein (A) (B) observes EGFP-PSLD with Hoechst dyeing nucleus, (C) nucleus is exposed to BrdU 1 hour, and be fixing then, with the antibody test of Cy-5 mark, with indication S phase cell.(D) the nucleus fluorescence intensity figure of the redness of the individual cells that exists in the complete visual field (the Cy-5 immunofluorescence of BrdU detects) and green (EGFP-PSLD).

Figure 11-pCORON1002-EGFP-C1-PSLD carrier collection of illustrative plates.

Figure 12-pCORON1002-EGFP-C1-β Gal-PSLD carrier collection of illustrative plates.

Figure 13-fluidic cell data has compared brightness and homogeneity with the signal of the representative stable cell lines of pCORON1002-EGFP-C1-PSLD, pCORON1002-EGFP-C1-β Gal-PSLD exploitation and parent U2OS clone.

Detailed Description Of The Invention

Method

Plasmid

By with total length HDHB cDNA as BglII/NotI fragment (Taneja et al., J.Biol.Chem., (2002) 277,40853-40861) be inserted into pEGFP-C1 carrier (Clontech, PaloAlto, NotI site CA) forms pGFP-HDHB and mutant derivative (seeing Fig. 4 and 6).(NotI site NY) makes up pFLAG-HDHB for Eastman Kodak Co., Rochester to be inserted into the pFlag-CMV2 carrier by the HindIII/NotI fragment that will contain total length HDHB cDNA.The structure of the HDHB-SLD of mark (1-1039) is by with the NruI after residue 1034 encoding sequences and with the NotI cracking mark HDHB plasmid in the poly joint, replaces small segment by flush end, terminator codon and outstanding 5 ' the compatible terminal double-end oligonucleotide of NotI that has the residue 1035 to 1039 of encoding.For forming pFLAG-HDHB (1-874), flat terminal with the pFLAG-HDHB DNA of Klenow polysaccharase treatment S tuI digestion to form, be connected in the pFLAG-CMV2 carrier.For forming pEGFP-β Gal, by the dna fragmentation of PCR, be inserted into 3 ' end of the GFP encoding sequence among the pEGFP-C1 by p β Gal-control (Clontech) amplification coding intestinal bacteria beta-galactosidase enzymess (β Gal), wherein use the HindIII site.The HDHB sequence of pcr amplification amino-acid residue 1040-1087 (SLD) and 957-1087 (PSLD) is inserted into the 3 ' end of the β GalcDNA among the pEGFP-β Gal, forms pGFP-β Gal-SLD and pGFP-β Gal-PSLD respectively.(La JoIIa CA) forms NES mutant and phosphorylation site mutant in HDHB cDNA for QuikChange, Stratagene by site-directed mutagenesis.

Make up pCORON1002-EGFP-C1-PSLD by pcr amplification 390bpPSLD zone from DNA construct pGFP-C1-β Gal-PSLD.5 ' NheI and 3 ' SalI restriction enzyme sites are incorporated in the PSLD fragment, make it possible to subclone to carrier pCORON1002-EGFP-C1 (GE Healthcare, Amersham, UK) in.Resulting 6704bp DNA construct pCORON1002-EGFP-C1-PSLD contains ubiquitin C promotor, bacterium ampicillin resistance gene and Mammals neomycin resistance gene (Figure 11).The nucleotide sequence of carrier is shown in SEQ ID No.3.Use standard clone technology (Sambrook, J.et al (1989)) forms three more forms of this carrier; At first replace the EGFP gene with J-Red (Evrogen), replace neomycin resistance gene, replace ubiquitin C promotor with CMV I/E promotor with hygromycin gene.

The structure of pCORON1002-EGFP-C1-β Gal-PSLD is by NheI and Xmal restriction enzyme digestion pEGFP-C1-β Gal-PSLD, and 4242bp EGFP-β Gal-PSLD fragment is inserted in the pCORON1002 carrier (GE Healthcare).Resulting 9937bpDNA construct pCORON1002-EGFP-C1-β Gal-PSLD (Figure 12) contains ubiquitin C promotor, bacterium ampicillin resistance gene and Mammals neomycin resistance gene.The nucleotide sequence of carrier is shown in SEQ ID No.4.

The protein of EGFP-PSLD fusion rotein and nucleotide sequence are shown in SEQ ID No.5 and 6 respectively.

Confirm the correct dna sequence dna of all constructs and replacement mutant by dna sequencing.

Antibody

Reorganization HDHB (Bethyl Laboratories, Montgomery, TX) the anti-HDHB antibody of generation, affinity purification (Harlow﹠amp on fixed HDHB at purifying; Lane, Antibodies:A laboratory manual.Cold Spring Harbor Laboratory).

Cell cultures, synchronization, microinjection, electroporation, transfection and stable cell lines form

The U2OS cell is being replenished 10% foetal calf serum (FBS) (Atlanta Biologicals, Norcross, GA) the improved Eagle substratum of Dulbecco (DMEM) (Gibco BRLLifetechnologies, Carlsbad, CA) in 37 ℃ of individual layers of cultivating the exponentiallies growth.By (incubation 24h makes the U2OS cell of exponential growth be stuck in G1/S among DMEM MO) for Sigma-Aldrich, St.Louis containing the 5mM thymidine.Interim for cell is discharged into S, the sucking-off substratum adds 10%FBS rinsing cell three times with the DMEM that heats, and adds incubation among the 10%FBS at fresh DMEM.U2OS cell with exponential growth in containing the DMEM that the 30ng/ml Lip river can reach azoles (Sigma-Aldrich) is stuck in G2/M 16h.Advance G1 for discharging cell, collect mitotic cell, add the 10%FBS rinsing three times, be tiled in then and be used for microinjection on the glass cover slide or be tiled in culture dish being used for further operation with DMEM by shaking out gently.

By foregoing flow cytometry (Taneja et al., J.Biol.Chem., (2002) 277,40853-40861) checking cell cycle synchronization.In the experiment of blocking-up nucleoprotein output, (Calbiochem, San Diego cultivate 3h and synthesize to stop new albumen among DMEM CA) at the leptomycin B (LMB) that contains 10ng/ml and 10 μ M cycloheximides with cell.(Herbig et al., the 1999) cell that will be tiled on the glass cover slide carries out microinjection as described, but injection is plasmid DNA rather than protein.

For electroporation, with the U2OS cell (5 * 10 of asynchronous growth ⁶) trypsinized, centrifugal collection is resuspended to the 20mM HEPES (pH 7.4) of 800 μ l, 0.7mMNa ₂HPO4/NaH ₂PO4,137mM NaCl, 5mM KCl, 6mM glucose, final pH 7.4.The DNA that adds 10 μ g, transfer to 0.4cm electroporation cup (BioRad, Hereules, CA) in, use Gene Pulser Il equipment (BioRad) to carry out electroporation.Cell is tiled in 1h in the tissue culture ware, uses the fresh culture rinsing, cultivate 23h again.

Work with the transient transfection cell proves, owing to low transfection efficiency, express the problem that high throughput analysis produced of inhomogeneous and these data, the porous flat plate form is difficult to.The influence of therefore screening a large amount of siRNA or reagent cell cycle just needs the generation of the stable cell lines of homogeneous.Because the toxic action during the long-term overexpression of HDHB has produced stable cell lines with the PSLD zone that is connected to reporter molecule.J-Red derivative transient transfection U2OS cell with pCORON1002-EGFP-C1-PSLD (Figure 11), pCORON1002-EGFP-C1-β Gal-PSLD (Figure 12) or above-mentioned carrier.Under suitable situation, use 1mg/ml G418 (Sigma) or Totomycin to select to express the stable clone of recombination fusion protein.By level and the homogeneity of the isolating former generation clone of flow cytometry (about 60 of each construct), and under suitable situation, use aforesaid method exploitation subculture clone to confirm that transmitter is expressed.

Fluorescent microscopy

For indirect IF staining, use phosphate buffered saline (PBS) (PBS) rinsing cell three times, with 3.7% formaldehyde fixed 20min among the PBS, saturatingization 5min in 0.2%Triton X-100, with the 10%FBS incubation 45min among the PBS.Add that with the anti-FLAG antibody of 1: 100 mouse monoclonal among the PBS (Sigma-Aldrich) 10%FBS detects FLAG-HDHB 2h in room temperature.After the rinsing, anti-(Jackson ImmunoResearch Laboratories, West Grove PA) add that 10%FBS is in room temperature incubation 1h with 1: 100 the red goat anti-mouse of Texas two of having puted together among the PBS.After three rinsings, with Hoechst 33258 (2 μ M are in PBS) incubation cell 10min.With cover glass be placed in ProLong Antifade (Molecular Probes, Eugene, OR) on.(Improvision, Lexington MA) go up the acquisition image in Zeiss Axioplan 2 imaging systems (Carl Zeiss Inc.) to use Openlab 3.0 softwares with the Hamamatsu digital camera.Cell count to the Subcellular Localization that shows every kind of pattern is counted, and is expressed as the per-cent of the total cellular score of being estimated (100 to 150 cells in each experiment).Every kind of proteinic ubcellular of qualitative assessment distributes at least two independent experiments.

For GFP fluorescence,,, carry out imaging and assessment as above-mentioned with the 3.7% formaldehyde fixed 20min that contains 2 μ M Hoechst 33258 with phosphate buffered saline (PBS) (PBS) rinsing cell three times.

Extract for Triton X-100, with cold cytoskeleton damping fluid (CSK, 10mMHEPES[pH 7.4], 300mM sucrose, 100mM NaCl, 3mM MgCl ₂) rinsing cell twice, extract 5min on ice with the 0.5%Triton X-100 in the CSK damping fluid (replenishing the 1X proteinase inhibitor), fixing as mentioned above then.

Under suitable situation, for the high-throughput imaging, dynamic imaging (24hr) in the porous flat plate form of stable cell lines fluorescent microscopy and analysis are to use high-throughput confocal imaging system (IN Cell Analyzer 1000 or IN Cell Analyzer 3000, GE Healthcare, Amersham, UK) in transfection carry out on the cell of red fluorescent protein (redFP) derivative of pCORON1002-EGFP-C1-PSLD, pCORON1002-EGFP-C1-β Gal-PSLD or these carriers.Use cell cycle phase markers algorithm (GEHealth Care) to analyze image.

The metabolic phosphate mark

With wild-type or mutant FLAGHDHB transient transfection U2OS cell (2.5 * 10 ⁶).Behind the 24h, with cell incubation 15min in removing phosphatic DMEM (Gibco BRL Lifetechnologies), with 32P-H3PO4 (0.35mCi/ml substratum; ICN PharmaceuticalsInc., Costa Mesa, CA) radio-labeled 4h.The FLAG-HDHB of immunoprecipitation phosphoric acid salt mark from extract separates through 7.5%SDS/PAGE, transfers on poly(vinylidene fluoride) as described below (PVDF) film.

Cell extraction, immunoprecipitation and westrern trace

24h after the transfection, the culture of the FLAG-HDHB transfection that desire is analyzed by immunoprecipitation and immunoblotting is at molten born of the same parents' damping fluid (50mM Tris-HCI pH 7.5,10% glycerine, 0.1%NP-40,1mM DTT, 25mM NaF, 100 μ g/ml PMSF, 1 μ g/ml presses down enzyme peptide, 1 μ g/ml leupeptin) molten born of the same parents in (0.5ml in each 35mm culture dish, perhaps 1ml in each 60mm culture dish or the 75cm flask).Wipe extract off from culture dish, at incubation 5min on ice, the centrifugal 10min of 14000g.With the anti-FLAG agaroses of 10 μ l (Sigma) on turner in 4 ℃ of incubation supernatant samples (0.5 to 1mg protein) 2h.With molten born of the same parents' damping fluid rinsing sepharose 4B three times.The protein transduction of immunoprecipitation is moved on on the pvdf membrane, with anti-HDHB peptide serum (1: 5000), anti-cell cyclin E antibody (1: 1000), anti-cell cyclin A antibody (1: 1000) (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and chemoluminescence agent (SuperSignal, Pierce Biotechnology Inc., Rockford is IL) through the western engram analysis.

Extract for selecting cell nuclear and cytoplasm protein,, use the PBS rinsing by the U2OS cell that tryptic digestion results 80-90% converges.With their resuspensions and at 10mMTris-HCl[pH 7.5], 10mM KCl, 1.5mM MgCl ₂, 0.25M sucrose, 10% glycerine, 75 μ g/ml digitonins, 1mM DTT, 10mM NaF, 1mM Na ₃VO ₄, 100 μ g/ml PMSF, 1 μ g/ml press down in enzyme peptide and the 1 μ g/ml leupeptin in molten born of the same parents 10min on ice, at the centrifugal 5min of 1000 * g.The supernatant liquor fraction is collected as the cytosol extract.To precipitate rinsing, be resuspended in high-salt buffer (10mM Tris-HCl[pH 7.5], 400mM NaCl, 1mM EDTA, 1mM EGTA, 1mM DTT, 1%NP-40,100 μ g/ml PMSF, 1 μ g/ml peptide presses down enzyme and 1 μ g/ml leupeptin) in, shake 10min at 4 ℃.After the supersound process, will contain solubility and analyze as the nucleus extract in conjunction with chromatinic proteinic suspended substance.By the protein in 8.5%SDS-PAGE analysis of cells nuclear and the tenuigenin extract, then the antibody with anti-α tubulin, PCNA (all from Santa Cruz Biotechnology) and reorganization HDHB carries out the wester trace.

The protein phosphatase reaction

30 ℃ with 100U in the Phosphoric acid esterase damping fluid (50mM Tris-HCl[pH 7.5], 0.1mM EDTA, 0.01%NP-40) (MA) incubation is attached to the FLAG-HDHB 1h of anti-FLAG pearl to the λ-Phosphoric acid esterase in for New England Biolabs, Beverly.There is or lacking inhibitors of phosphatases (5mM Na ₃VO ₄, 50mM NaF) situation under react.By 7.5%SDSPAGE (acrylamide-bisacrylamide ratio, 30: 0.36) isolated protein, detect HDHB through the western trace with anti-HDHB serum and chemoluminescence.

The peptide mapping of trypsin hydrolyzing and phosphorylated amino acid assay

24h after the transfection, as above handle the culture of the radiolabeled FLAG-HDHB transfection that will be used for immunoprecipitation and phosphoamino acid or phospho-peptide mapping, but with the RIPA damping fluid (50mM Tris-HCl[pH 7.5], 150mM NaCl, 1%NP-40,0.5% Septochol, 1%SDS, 50mM NaF, 1mM EDTA, 5mM Na ₃VO ₄, 100 μ g/ml PMSF, 1 μ g/ml presses down enzyme peptide and 1 μ g/ml leupeptin) the molten born of the same parents' damping fluid of replacement.Through the sedimentary protein of 7.5%SDS-PAGE separating immune and transfer on the pvdf membrane.Fully wash the film twice that contains radiolabeled HDHB with deionized water, then through autoradiography observation phosphoric acid protein.Then phosphoric acid protein is downcut, follow water wetting film fragment again with methyl alcohol.With the 50mM NH that contains 0.1%Tween 20 (Sigma-Aldrich) ₄HCO ₃At room temperature closing membrane 30min, use 50mM NH ₄HCO ₃Rinsing three times, the ox pancreas trypsin Worthington that uses L-(tolylsulfonyl amido-2-phenyl) ethyl chloride methyl ketone to handle then, Lakewood NJ) goes up enzyme with phosphoric acid protein from PVDF and scales off.Then as other local detailed description in detail like that (Boyleet al., Meth.Enzymology, (1991), 201,110-149) the two-dimentional phospho-peptide that carries out peptide is mapped or the phosphorylated amino acid assay.

The reaction of cyclin dependant vitro kinase

Cyclin/the CDK (200pmol/h) (providing by R.Ott and C.Voitenleitner) of purifying and reorganization HDHB (the Taneja et al. of purifying are provided, J.Biol.Chem., (2002) 277,40853-40861) as substrate according to former described kinase reaction (the Voitenleitner et al. that carries out like that, MoI.Cell.Biol., (1999), 19,646-56).

The BrdU mark, in the chemical cell cycle blocking-up that stabilized cell is fastened and the affirmation of RNAi experiment

Use the substratum (100 μ l/ hole) of antibiotic-free will express the stabilized cell of pCORON1002-EGFP-C1-PSLD construct with 0.3 * 10 ⁵/ ml is seeded in the 96 hole Greiner flat boards, incubation 16 hours.

For confirming EGFP-PSLD, use cell amplification test kit (Amersham Biosciences, GE Health Care) BrdU mark stabilized cell 1hr in the interim distribution of S.Cell is fixing in 2% formalin, with second antibody system (the cell amplification test kit of Cy-5 mark; GE Health Care) detects the BrdU that mixes by immunofluorescence.Examine with hoechst (2 μ M) staining cell.

For chemical block research (table 1), stabilized cell is exposed to olomoucine, roscovitine, Luo Keda azoles, mimosine, Omaine or colchicine (Sigma).Cell is fixing in 2% formalin, with hoechst (2 μ M) staining cell nuclear.

Study for siRNA, to resist the siRNA set (Dharmacon) of some cyclin, MCM albumen, CDK, polo sample kinases (PLK) and contrast duplex (table 2) at random in lipofectamine/optimem I (Invitrogen), to be diluted to 25nM, join 4hr in the stabilized cell.Replace substratum, the dull and stereotyped 48hr of incubation.Cell is fixing in 2% formalin, with hoechst (2 μ M) staining cell nuclear.

In IN Cell Analyzer system (GEHC), carry out after high-throughput imaging and the analysis, use hoescht to obtain the data of the average core density and the N of the individual cells sum in the visual field: C as nucleus mark and IN Cell Analyzer 3000 cell cycle phase markers algorithms (GEHC) than the nuclear signal intensity (BrdU) under (EGFP signal), nuclear size (hoescht signal) and the suitable situation.Concerning each hole, it (mainly is that nuclear EGFP distributes that the total cellular score in each visual field is categorized into the G1 phase; High EGFP-PSLD nuclear density and N: C ratio), S phase (3SD on nuclear BrdU signal＞background; EGFP-PSLD N: C is than about 1) and G2 phase (bigger nuclear size; Lower EGFP-PSLD N: C ratio).Although it is possible distinguishing M phase cell (according to less nuclear size and very strong EGFP signal), in the hole of formalin fixed, seldom see such cell, in rinsing and fixation procedure because they have been removed.

The result

HDHB is present in epipole or the tenuigenin

For determining the Subcellular Localization of endogenous HDHB, selective extraction nuclear and cytoplasm protein separate by denaturing gel electrophoresis, by western engram analysis (Fig. 1) from people U2OS cell.At first monitor the PCNA in every kind of extract and the existence of alpha-tubulin and assess extraction procedure.PCNA is enrichment in nuclear extract, and not enrichment in the tenuigenin fraction, and alpha-tubulin mainly is found in the tenuigenin fraction, has confirmed fractional separation.In nuclear and tenuigenin fraction, all detected HDHB (Fig. 1).Tenuigenin HDHB moves slower (Fig. 1) than the nuclear fraction, has hinted the possibility of posttranslational modification.

These the possibility of result show or HDHB is distributed in the whole cell, or mixed cellularity group all contains HDHB in nucleus or tenuigenin.For distinguishing these options, with HDHB original position location in individual cells; In people U2OS cell, express the HDHB of GFP and FLAG mark by transient transfection.Because the long-term overexpression of mark or unmarked HDHB is Cytotoxic, all experiments are all carried out (24h usually) in possible short duration.The mark HDHB that analyzes in the individual cells by fluorescent microscopy locatees.GFP-HDHB and FLAG-HDHB have shown two kinds of main station-keeping modes, or in nucleus in the dispersive epipole, or in tenuigenin (Fig. 1).In nucleus or tenuigenin, also all observed the transient expression of GFP-HDHB in former generation human fibroblasts.

The affirmation of cell cycle dependency Subcellular Localization structural domain among the HDHB

With the Luo Keda azoles U2OS cell is stuck in G2/M, was discharged into G1 three hours, use pGFP-HDHB DNA microinjection then in their nucleus.The expression of GFP-HDHB is easy to detect after six hours, and the G1 phase cell of this moment about 70% is mainly at nucleus accumulation fusion rotein (Fig. 2).On the contrary,, use pGFP-HDHB DNA microinjection then, surpass 70% S phase cell and mainly in tenuigenin, accumulate fusion rotein (Fig. 2) when cell synchronization during at G1/S, being discharged into the S phase with thymidine.The selective extraction of G1 and S phase U2OS cell shows that endogenous HDHB major part is at nuclear and the tenuigenin of S phase (Fig. 2 b) of G1.Yet endogenous HDHB can detect in two kinds of subcellular fractions significantly.Compare with G1 phase albumen, the mobility of S phase HDHB has slightly been delayed.These results show that the Subcellular Localization of HDHB is regulated in the cell cycle, the HDHB of GFP mark has reflected the location of endogenous unmarked helicase.

Be subjected to Bloom Cotard helicase and the helicase (Hickson of other RecQ family, Nature Rev.Cancer, (2003) 3,169-178) in the prompting differentiated of C-terminal nuclear localization signal, possible Subcellular Localization structural domain (SLD) is differentiated C-terminal (Fig. 3) at HDHB.For the HDHB truncated mutant (GFP-HDHB-.SLD) determining whether this SLD that infers important for the HDHB location, prepared to lack 48 residues of C-terminal that contain SLD (Fig. 3).The expression vector microinjection in G1 or S phase U2OS cell, is checked the Subcellular Localization of fusion rotein by fluorescent microscopy after six hours.Surpass 95% cell and in tenuigenin, accumulated fusion rotein, no matter the cell cycle time that HDHB expresses how (Fig. 3 c).This result shows that HDHB may have NLS, its in GFP-HDHB-Δ SLD by C-terminal disappearance and weakened or eliminated.

For the C-terminal structural domain of determining HDHB whether enough for appraising and deciding the position, use bacteria beta-galactosidase (β Gal) as report albumen, because it has the molecular weight (112kDa) near HDHB, and do not contain Subcellular Localization signal (Kalderon et al., Cell, (1984), 39,499-509).In contrast, preparation GFP-β Gal expression vector (Fig. 3) is with the Subcellular Localization of expression vector microinjection monitoring fusion rotein behind the U2OS cell.As expected, GFP-β Gal mainly is accumulated in (Fig. 4) in the tenuigenin.On the contrary, in the nucleus of asynchronous or synchronized U2OS cell and tenuigenin, all found GFP-β Gal-SLD (Fig. 4), shown that SLD contains NLS, but proteic to appraise and decide the position be not enough for report.Infer perhaps contiguous potential CDK phosphorylation site and may influence the Subcellular Localization in the cell cycle (Fig. 3), just made up GFP-β Gal-PSLD, 131 residues of C-terminal that wherein will contain the HDHB of the SLD that infers and potential CDK phosphorylation site bunch append to the C-terminal (Fig. 3) of GFP-β Gal.When GFP-β Gal-PSLD plasmid DNA in asynchronous and synchronous U2OS cell during transient expression, in surpassing the nucleus of 90% G1 phase cell and surpass in the tenuigenin of 70% S phase cell and found GFP-β Gal-PSLD (Fig. 4).With the observed confocal pattern of the nuclear GFP-HDHB among the G1 is compared, uniform distribution in GFP-β Gal-PSLD and the EGFP-PSLD albumen whole nucleus in G1 is a spot of at kernel only.To having emphasized with the analysis of the stable cell lines of the expression pCORON1002-EGFP-C1-PSLD of BrdU mark that S phase cell (equaling about 60% asynchronous group) shows the uniform distribution of EGFP-PSLD signal or mainly at cytoplasmic distribution (Figure 10).S phase cell does not show that the main nuclear of the EGFP-PSLD relevant with the G1 cell distributes.Find that some cells demonstrate the absolute kernel repulsion (Figure 10) of EGFP-PSLD reporter molecule, yet these cells do not mix BrdU.We suppose that the absolute cell of removing EGFP-PSLD is G2 in the showed cell nuclear.The dynamic imaging that surpasses 24 hours EGFP-PSLD stable cell lines demonstrates EGFP-PSLD mainly in the nuclear of the G1 after mitotic division, shown G1/S change before and after (after the division of cytoplasm about 3.5 hours) move by examining tenuigenin rapidly, and translocate to tenuigenin from nucleus from G1/S gradually up to G2 latter stage (about 19 hours) further; At this moment, before the division cell rounding is taking place once more.As if these observationss confirmed that the G2 cell demonstrates the absolute tenuigenin distribution of EGFP-PSLD reporter molecule.When comparing, find that the stable cell lines of expression EGFP-PSLD fusions influences the length overall (about 24 hours) of cell cycle with U2OS cell or G2M cell cycle phase markers clone (GEHC).In a word, these data show that the Subcellular Localization of HDHB depends on the cell cycle, the C-terminal PSLD structural domain of HDHB plays main effect aspect the proteinic Subcellular Localization regulating in cell cycle dependence mode, HDHB in the nuclear of G1, but in the S phase with translocate to tenuigenin gradually during the G2 possibly.

The discriminating of functional rev type NES among the HDHB

The verified numerous protein that shuttles back and forth between nucleus and tenuigenin comprises the NES (Fig. 5) that is similar to the proteic prototype NES of HIV rev.The protein that contains rev type NES need be exported factor CRM1 (being also referred to as nuclear output albumen 1) and remove conjugated protein and it is transported to tenuigenin (by Weis, Cell, (2003), 112,441-451 comments) from nucleus.Leptomycin B (LMB) specificity suppresses CRMl activity (Wolff etal., Chem.Biol., (1997), 4, the 139-147 in the nucleoprotein output; Kudo et al., Exp.Cell.Res., (1998), 242,540-547).Inspection to the PSLD sequence among the HDHB has shown the rev type NES (LxxxLxxLxL that infers; Fig. 5).For whether the tenuigenin location of determining HDHB needs functional NES, the expression plasmid of GFP-HDHB or FLAG-HDHB DNA is existed or lacking under the situation of LMB microinjection in nonsynchronous, G1 and S phase cell.Check the location of fusion rotein by fluorescent microscopy, and carry out quantitatively.When having LMB, two kinds of fusion roteins all are accumulated in the nucleus, and with cell cycle irrelevant (Fig. 5), this is consistent with the possibility that HDHB contains the rev type NES that works by CRMl.Yet also possible is, HDHB may not be CRMl directly transport thing, its output may be by the mediation indirectly of some other protein.For whether the NES that infers among the assessment HDHB is functional, we are mutated into L-Ala to produce NES mutant 1 and 2 (Fig. 5) with the Val/Leu and the Leu/Leu of NES motif.The GFP-HDHB and the GFP-β Gal-PSLD transient expression in asynchronous or synchronized U2OS cell that comprise these NES mutant.Two kinds of NES sudden change fusion roteins all are accumulated in the nucleus in surpassing 80% cell, and no matter when they express (Fig. 5) in asynchronous or synchronization cell.This result shows that NES sudden change specificity has weakened the output of GFP-HDHB and GFP-β Gal-PSLD, proves that functional NES is contained in the PSLD zone of HDHB.

FLAG-HDHB in vivo in cell cycle dependency mode by phosphorylation.

Potential CDK phosphorylation site bunch (Fig. 3) in the PSLD structural domain of HDHB shows that the phosphorylation of HDHB may be regulated its Subcellular Localization in the cell cycle.If like this, people will expect the PSLD zone of HDHB in cell cycle dependency mode by phosphorylation.Whether HDHB experiences phosphorylation among the PSLD for test, with the wild-type of FLAG-HDHB and the expression plasmid transient transfection U2OS cell of C-terminal clipped form, use the phosphoric acid salt radio-labeled, then FLAG-HDHB immunoprecipitation from cell extract is come out, by the protein (Fig. 6) of denaturing gel electrophoresis, immunoblotting and radioautographic analysis immunoprecipitation.Detected the radio-labeled band (Fig. 6 A, swimming lane 1) of FLAG-HDHB at the same position of immunocompetence HDHB band.The brachymemma FLAG-HDHB that lacks SLD also in vivo by strong phosphorylation (swimming lane 2), and the brachymemma FLAG-HDHB (1-874) that lacks PSLD is not by remarkable phosphorylation (swimming lane 3).These results show that SLD is not that the HDHDB phosphorylation is necessary, and PSLD is essential, and show that phosphorylation site may be positioned at PSLD.

For checking the HDHB time of phosphorylation in the cell cycle, not using radio-labeled to detect phosphorylation will be easily.Because phosphorylation has often reduced the electrophoretic mobility of protein in denaturant gel, with the FLAG-HDHB immunoprecipitation of transient expression, with λ-Phosphoric acid esterase (λ-Ppase) handle before and check its mobility (Fig. 6 B) afterwards.When not having λ-Ppase to handle, in the western trace, detected FLAG-HDHB (swimming lane 1) in two very approaching migration bands, and the FLAG-HDHB of phosphorylation moves (swimming lane 2) as single band with the mobility of the very fast band of two bands.When having λ-Ppase inhibitor in the reaction, FLAG-HDHB moves (swimming lane 3) as the identical two bands of the protein of handling with vacation.These data show that the electrophoretic mobility of FLAG-HDHB has been reduced by phosphorylation, and this analytical method may be applicable to follows the trail of the phosphorylation of HDHB in the cell cycle.

For determining that whether HDHB relies on mode by phosphorylation with the cell cycle, the U2OS cell of transient expression FLAG-HDHB is stuck in G1/S or in substratum it is stuck in G2/M by adding the Luo Keda azoles in the substratum by adding thymidine.Cell is discharged the different time periods from blocking-up, immunoprecipitation goes out FLAG-HDHB from cell extract.

With or without λ-Ppase incubation immunoprecipitate, then by denaturing gel electrophoresis and western engram analysis (Fig. 6 C).Increased from stagnating to be handled by λ-Ppase in the FLAG-HDHB mobility of the cell of G1/S, show protein at G1/S by phosphorylation (Fig. 6 C, last figure).After discharging from the G1/S blocking-up, Phosphoric acid esterase is handled FLAG-HDHB and has been detected similar mobility change (last figure) at least after nine hours, also is (Fig. 6 C, figure below) blocking in the cell of G2/M.Yet, being released among the G1 after four and eight hours at cell, FLAG-HDHB is as single band migration, and the influence that handled by Phosphoric acid esterase is just littler (Fig. 6 C, figure below).After discharging from G2/M blocking-up by 12 hours, when most cells is just entering S during the phase (data not shown), the mobility of FLAG-HDHB is handled by Phosphoric acid esterase again to be increased, and had recovered viewed pattern (figure below) in the cell that the Luo Keda azoles stagnates.These results show that strongly the phosphorylation of FLAG-HDHB is the cell cycle dependency, from G1/S maximum phosphorylation during G2/M, minimum phosphorylation during G1.

Serine 967 is the main phosphorylation sites of the HDHB of ectopic expression

For drawing the phosphorylation site collection of illustrative plates among the FLAG-HDHB, we wish at first to determine which amino-acid residue has been modified.The phosphorylated amino acid assay of radiolabeled FLAG-HDHB shows that phosphoserine is a FLAG-HDHB main phosphoamino acid (Fig. 7 A) in vivo in the body.Suppose among the PSLD of cell cycle dependency phosphorylation site between residue 874 and 1039 of HDHB (Fig. 3 A), these sites are modified by CDK, phosphoserine is adorned main amino acid (Fig. 7 A), so seven potential CDK sites have only four will be still as candidate locus.Have the FLAG-HDHB expression plasmid that corresponding Serine becomes the sudden change of L-Ala for testing each these site separately, having made up.To use the orthophosphoric acid salt radio-labeled in vivo with the cell of these plasmid transient transfections,, analyze (Fig. 7 B) by radioautograph and western trace with the FLAG-HDHB immunoprecipitation.These results show that FLAG-HDHB and three kinds of mutains are by approximate phosphorylation comparably, and the S967A mutain is only by weak phosphorylation (Fig. 7 B).This result's hint, S967 may be the main site of phosphorylation in the HDHB body.Consistent with this explanation, the electrophoretic mobility of FLAG-HDHB after Phosphoric acid esterase is handled that has detected immunoprecipitation with three kinds of mutains changes, but do not detect with S967A albumen.

For confirming that S967 is a phosphorylation site in the main body of HDHB, carry out the phospho-peptide drawing of trypsin hydrolyzing with wild-type and S967A sudden change FLAG-HDHB, they have used orthophosphoric acid salt metabolic ground radio-labeled (Fig. 7 C).Observed the peptide (left figure) of the radiolabeled peptide of a kind of advantage and a kind of weak mark with wild-type protein.The phospho-peptide that in S967A albumen, lacks the advantage mark, but the peptide (Fig. 7 C, right figure) of mark a little less than still can detecting.These results provide extra evidence, prove that Serine 967 is the intravital remarkable phosphorylation sites of HDHB.

Cyclin E/CDK2 is as the kinase whose affirmation of HDHB among the potential modification G1/S

In fact can CDK modify HDHB for test, as by the HDHB phosphorylation time in the cell cycle and S967 as suggested in the affirmation in the main site of modifying, with the reorganization HDHB of purifying and the cyclin E/CDK2 or the cyclin A/CDK2 of the external incubation purifying of radio-labeled ATP.After kinase reaction, by the denaturing gel electrophoresis isolated protein, transfer on the pvdf membrane, detect by radioautograph.The result shows that reorganization HDHB can be by cyclin E/CDK2 and the strong phosphorylation of cyclin A/CDK2.Further radiolabeled HDHB band is processed then, the phospho-peptide that is used for trypsin hydrolyzing is drawn.With the peptide two dimensional separation of every kind of digestion, or separate separately, otherwise with body in the trypsin hydrolyzing peptide of phosphorylation FLAG-HDHB separate after mixing, by autoradiography observation (Fig. 8 A).By the HDHB peptide of cyclin E/CDK2 and cyclin A/CDK2 phosphorylation produced with body internal labeling peptide mapping in viewed essentially identical pattern, a main spot and one are arranged than speckle (figure A).When outside the mixture with the peptide of body internal labeling and when on a kind of tomographic map, separating, their are migration (Fig. 8 A, right side) altogether.These digital proofs are modify in by body among the FLAG-HDHB same a kind of by the main phospho-peptide of cyclin E/CDK2 and the external modification of cyclin A/CDK2 in the reorganization HDHB of purifying.

Because the cyclin E activity in people's cell took place in the late G1 phase, and cyclin A activity is (Pines, 1999 that occur generation subsequently with the S phase; Erlandssonet al., 2000), so attempt distinguishing that whether one of these kinases can preferentially modify HDHB is important.The cyclin subunit often forms the mixture with the substrate protein white matter, their target phosphorylations (Endicott et al., 1999; Takeda et al., 2001).For whether test cell cyclin E or cyclin A can associate with HDHB, from transfection immunoprecipitation FLAG-HDHB and associating protein the cell extract of FLAG-HDHB expression vector or empty FLAG carrier in contrast.By western engram analysis cell extract and immunoprecipitate (Fig. 8 B).Cyclin E obviously and the FLAG-HDHB co-precipitation, but cyclin A (Fig. 8 B, swimming lane 2 and 5) not like this shows that FLAG-HDHB may preferentially interact with cyclin E in vivo.This interaction may be that HDHB is necessary by phosphorylation in the cyclin E/CDK2 body, this is conceivable, if like this, prevent among the HDHB that it and the associating sudden change of cyclin E from will end the phosphorylation that cyclin E/CDK2 is carried out.For testing FLAG-HDHB mutant S967A because can not be in conjunction with cyclin E in vivo not by the possibility of phosphorylation (Fig. 7 B, C), immunoprecipitation FLAG-HDHB-S967A and associating protein are analyzed by the western trace from the transfectional cell extract.Cyclin E is the same with wild-type FLAG-HDHB with the co-precipitation of mutain strong.

It is critical that the phosphorylation of Serine 967 is located for control HDHB.

The Subcellular Localization of the HDHB of above-mentioned data presentation ectopic expression and phosphorylation are controlled in cell cycle dependency mode, have maximum phosphorylation from G1/S to G2/M, and this is consistent with the period that HDHB accumulates in the tenuigenin.These results and S967 show that the phosphorylation of S967 may regulate the Subcellular Localization of HDHB as the affirmation of phosphorylation site in the main body among the HDHB together.For testing this idea, with the expression plasmid microinjection of wild-type GFP-HDHB and mutant S967A, S984A, S1005A and S1021A in synchronized U2OS cell.As expected, wild-type GFP-HDHB is accumulated in the epipole of G1 cell, but in the tenuigenin of S phase cell.Yet regardless of cycle time, GFP-HDHB-S967A is positioned the epipole (Fig. 9) of about 70% fluorocyte.Other three kinds replace mutant or are positioned nucleus, or resemble and be positioned in the tenuigenin the wild-type GFP-HDHB.In the effort of attempting simulation S967 phosphorylation, Serine 967 is mutated into aspartic acid, in nonsynchronous and synchronized U2OS cell, express GFP-HDHB-S967D, check that the ubcellular of sudden change fusion rotein distributes.

About 60% the cell of expressing GFP-HDHB-S967D has shown tenuigenin fluorescence (Fig. 9 A) in nonsynchronous, G1 phase and S phase cell, prove the S967 that S967D suddenlys change and simulated phosphorylation.These data have shown that effectively the phosphorylation of Serine 967 is being critical aspect the Subcellular Localization of regulating HDHB.

The C-terminal structural domain of HDHB has been given cell cycle dependency location

The structural domain PSLD of 131 residues enough examines HDHB, EGFP or β Gal reporter molecule or tenuigenin (Fig. 4 and 10) with cell cycle dependency mode targeted cells.Rev-type NES is arranged in this structural domain (Fig. 5), but it the active or accessibility of nuclear output mechanism is depended on PSLD mainly is the phosphorylation (Fig. 6-9) of Serine 967 when G1/S changes.The S967 Perfect Matchings is in total CDK substrate identification motif (S/T) PX (K/R).Cyclin E/CDK2 and cyclin A/CDK2 both can external modification HDHB, but the HDHB compound ability in cyclin E/CDK2 and the cell extract shows that it may be an initial kinases (Fig. 8) of modifying HDHB when G1/S changes.For the EGFP-PSLD stable cell lines, the adding (table 1) of known Cdk2 inhibitor olomoucine and roscovitin or caused EGFP-PSLD mainly to distribute and be stuck in G1 at nuclear at the adding (table 2) of the siRNA of cyclin E, this has further supported Cdk2/ cyclin E to be responsible for controlling the possibility of viewed phosphorylation dependency Subcellular Localization based on the cell cycle.The phosphorylation of PSLD appear to last till the cell cycle than the rear section, this mainly is that the tenuigenin location is very relevant in S and G2 with HDHB.Handling the kinetics image of stable cell lines above 24 hours with olomoucine shows, for the cell that is stuck in G2, the EGFP-PSLD signal redistributed nucleus from tenuigenin and surpasses (not having cell by mitotic division) in about 4-8 hour, this shows and is lacking under the active situation of cdk2, EGFP-PSLD otherwise become dephosphorylation and reenter nucleus, destroyed and new synthetic protein because cdk2 suppresses not by phosphorylation, thereby be arranged in nucleus.

Table 1

Compound	％			Total cell
					S	G1	G2
	Omaine (0.3 μ M)	41	16					43	490
Omaine (1.2 μ M)				32	8	59	450
	Colchicine (4 μ M)	36	9					55	467
Colchicine (100 μ M)				32	12	57	439
	L-mimosine (2mM)	68	6					26	1710
Olomoucine(500μM)				33	63	4	600
	Roscovitin(100μM)	36	52					13	693
Luo Keda azoles (3 μ M)				33	6	61	606
	Contrast	61	17					22	2137

Table 2

siRNA	％			Total cell
					S	G1	G2
	PLK	53	9					38	66
MCM7				58	13	29	231
	MCM6	64	14					22	166
MCM5				63	17	20	260
	MCM4	56	20					24	223
MCM3				59	23	19	188
	MCM2	50	24					26	266
Cell periodic protein B 1 V2				49	36	15	280
	Cell periodic protein B 1 V1	60	24					17	203
CDK8				50	23	27	299
	CDK7	56	18					26	354
CDK6				58	22	20	328
	Cyclin A2	61	13					26	319
Cyclin A1				66	10	24	298
	Cyclin T2b	57	12					31	267
Cyclin T2b				55	22	23	355
	Cyclin T1	60	20					20	260
Cyclin E1				49	27	24	272
	Contrast	69	10					20	262

Can not distinguish whether HDHB has experienced dephosphorylation (Fig. 6 C) or may have been carried out proteolysis and synthetic again rapidly in early days at G1 by target when M/G1 changes, it will enter nucleus at that time.Yet, the stable cell lines kinetics imaging that surpasses 24 hours shows, the EGFP-PSLD signal during the M phase or M/G1 when boundary not by very big reduction, but suffered (this state continues about 3 hours during G1 then) at nuclear just becoming mainly in about 30 minutes after the division of cytoplasm, this is consistent with nuclear membrane formation.This shows the EGFP-PSLD construct by dephosphorylation, rather than experience is significantly destroyed before and after the M/G1 boundary.

These data provide strong evidence, prove that PSLD contains the active targeting signal (Fig. 2-5,10) that does not rely on the protein environment.Even since the sudden change HDHB that has an inactivation NES when it was expressed during the S phase also in nuclear, so presumably be exactly by phosphorylation (Fig. 5), possible NES is not by the deactivation of phosphorylation institute, the major objective that CDK controls is NES.Extend this reasoning, NES may be masked during G1, and this moment, the CDK motif among the PSLD was not modified, when S967 during by phosphorylation NES just be released, caused NES identification (Fig. 3-5) through the nuclear output factor.Structural research to rev type NES has shown that it has formed amphipathic alpha-helix, and leucine is arranged on the side of spiral, charged residue be arranged in opposite side (Rittinger et al., MoI.Cell.Biol. (1999), 4,153-166).Because the SLD of HDHB contains rev type NES and NLS, may be dispersed in as the alkaline residue that NLS works among the NES, NES and NLS may be positioned at the opposite face of amphipathic helix.Additional sequences among the PSLD will be sheltered NES at intramolecularly, and this makes to have only NLS just can be identified.The phosphorylation of S967 will change shelters conformation exposing NES among the PSLD, and does not influence the exposure of NLS.

Come the inhibitor of high flux screening cell cycle with the EGFP-PSLD stable cell lines

As mentioned above, the of service that carries out with the transient transfection cell is understood the difficulty of porous flat plate form, and this is owing to low transfection efficiency, express the problem that high throughput analysis produced of inhomogeneous and these data.Therefore just need the generation of the stable cell lines of homogeneous for the screening of a large amount of siRNA or the influence of reagent cell cycle.Produce stable cell lines with the PSLD zone that is connected on the reporter molecule (EGFP), described connection is by the joint of the seven amino acid of flexibility (using pCORON1002-EGFP-C1-PSLD).As can be seen from Figure 13, the fluorescent signal of being set up with pCORON1002-EGFP-C1-β Gal-PSLD that stable cell lines produced is than the signal that clone produced obviously smaller (about ten times) of the joint with flexible seven amino acid.This may be to need transcribing and translating mechanism of cell owing to the proteic size of β Gal morely.

The stable cell lines of setting up with pCORON1002-EGFP-C1-PSLD (seeing Figure 13) be in itself homogeneous (average total cell RFU 435, SD 58; N=271; See Figure 10), provide sensitive, stable and consistent analytical method to be used for the porous flat plate form research cell cycle and the influence (table 1 and 2 of screening reagent cell cycle rapidly; Figure 10).

Above disclosed some aspect of the present invention is at Molecular Biology of theCell (15:3320-3332, in July, 2004) disclose in, and on May 14th, 2004 electronics be disclosed in MBC to be delivered, 10.1091/mbc.E04-03-0227, exercise question is " CellCycle-dependent Regulation of a Human DNA Helicase That Localizesin DNA Damage Foci ", with the whole introducing of these disclosures here as a reference.

Foregoing is explanation the present invention, is not interpreted as the restriction to it.Although described illustrative embodiments more of the present invention, what those skilled in the art will be readily appreciated that is that it is possible not deviating from new instruction of the present invention and many modifications of advantage in the illustrative embodiment in fact.Therefore, these all modifications all are confirmed as comprising within the scope of the invention, as defined in the claim.So it being understood that foregoing is explanation the present invention, is not interpreted as to be limited to disclosed particular, and modification and other embodiment of disclosed embodiment all is confirmed as comprising within the scope of the appended claims.The present invention limits by following claim, and the equivalent of claim is included in wherein.

Sequence table

SEQ ID NO：1

atggccaggt cgagtccgta cctgcgccaa cttcagggac ctctgctccc acccagggat 60

ctggtggagg aggacgacga ctacctaaac gacgacgtgg aggaggatga agagtccgtg 120

ttcatcgacg ccgaggagct ctgcagtggg ggcgtaaagg ctggcagcct ccccgggtgc 180

ctccgcgttt ctatttgtga tgaaaacaca caagagacat gtaaagtgtt tggacgtttt 240

ccgataacag gtgcttggtg gagagtgaag gtacaagtaa agcctgtggt gggatcaagg 300

agctatcaat atcaagttca aggatttccg tcttactttt tgcagtctga tatgtcacca 360

ccaaatcaaa aacatatctg tgctctcttt cttaaagagt gtgaggtctc cagtgatgat 420

gttaataaat ttttaacatg ggtaaaggag gtatcaaact acaaaaacct aaactttgaa 480

aatcttaggg aaacactaag aactttccac aaggaaactg gaaggaaaga tcaaaagcag 540

cctacacaga atggtcagga agagttgttc ctagacaatg agatgagtct tcctctggaa 600

aacacaattc catttagaaa tgtaatgaca gctttgcagt ttccgaagat aatggaattc 660

cttccagttc ttctgcctcg acactttaaa tggatcatag ggtcaggttc taaagagatg 720

ttgaaagaga tagaagagat tttaggtaca catccgtgga aacttggatt tagtaaaata 780

acctacagag agtggaaact cctgcgatgt gaggcaagtt ggatagcatt ttgtcagtgt 840

gagtctcttc tccagctgat gactgatttg gagaagaatg cattaataat gtattccaga 900

ctgaagcaga tatgtagaga agatgggcac acatatgttg aagtgaatga cttaactttg 960

acattgtcaa atcatatgtc atttcatgct gcttcagagt ctctgaagtt tttgaaggat 1020

attggtgtgg tgacatatga gaagtcctgt gtcttccctt atgaccttta ccatgctgaa 1080

agagccatcg ccttttcaat ttgtgacctg atgaagaaac ctccttggca tttatgtgtc 1140

gatgtcgaaa aggtgcttgc ctctattcac accacaaaac ctgagaattc aagcgatgat 1200

gcattgaatg agagcaaacc tgatgaagta agattagaaa atcctgtgga tgttgtggac 1260

acacaggaca atggtgacca tatttggact aatggtgaaa atgaaattaa tgcagaaata 1320

agtgaagttc agctggatca ggatcaggtt gaagttccac tggatcggga tcaggtggct 1380

gctttggaaa tgatttgctc caatcctgtg acagtcataa gtgggaaagg tggatgtggg 1440

aagaccacaa tcgttagccg tctttttaag catatagagc agttggaaga aagagaagta 1500

aaaaaagcct gtgaagattt tgaacaagac cagaatgctt cagaagaatg gattaccttt 1560

actgagcaaa gtcaactaga ggcggacaag gctatagaag ttttgctcac agcacctaca 1620

gggaaagcag ctggcttact aagacagaaa actggtcttc atgcctacac actgtgtcag 1680

gtcaattata gcttctattc atggactcaa acaatgatga ccacaaacaa accatggaaa 1740

ttttcttcgg ttagagttct ggttgtggat gaagggagtt tggtatctgt aggaatcttc 1800

aaatcggtct taaatttatt gtgtgagcac tccaaacttt ctaagcttat tatccttggt 1860

gacattagac agttacccag tattgaacct ggtaacttgc tgaaagatct ttttgagact 1920

cttaagtcaa gaaattgtgc tattgagcta aagacaaacc atagagcaga atctcagctc 1980

attgtggaca atgctacaag aatctcaaga cgccaatttc caaaatttga tgcagaacta 2040

aatatctctg ataatccaac attacccatc tcaattcaag ataagacatt tatttttgtc 2100

aggctcccag aagaggatgc cagttctcag tcatctaaaa ctaatcatca ctcttgttta 2160

tattctgcag ttaaaacttt actacaagaa aataacttac aaaatgcaaa aacatcacaa 2220

tttattgcat ttagaaggca agactgtgat ctaattaatg actgctgctg caaacactac 2280

acaggccacc tcaccaaaga ccatcagagt agacttgttt ttggaattgg tgataaaatt 2340

tgttgtacca ggaatgcata cctctcagac ttactacctg aaaatatctc tggaagtcag 2400

caaaataatg atctagatgc cagtagtgaa gacttttctg gtacgcttcc tgattttgct 2460

aaaaataagc gtgactttga aagtaacgtt cgactgtgca atggagagat atttttcata 2520

acaaatgatg taactgatgt aacttttgga aagagaagat ctttgaccat taataatatg 2580

gctggcctgg aagtaactgt ggattttaag aaactaatga aatattgtcg cataaaacat 2640

gcatgggcaa gaactattca cacttttcag gggtccgagg agcaaacagt tgtctatgtg 2700

gtggggaagg cgggccgcca gcactggcag catgtctaca ccgccgtgac caggggccgc 2760

tgccgagtgt atgtgattgc agaggagtct cagctccgga atgccattat gaaaaacagt 2820

tttcctagaa aaactcgttt gaaacatttc ttgcaaagta agctctcctc tagcggcgca 2880

cctccagcag attttccgtc cccacggaag agctctggag acagtggagg acccagcaca 2940

ccgtcagcat ctccactccc tgtagtcaca gaccacgcca tgacaaatga tgtcacctgg 3000

agcgaggcct cttcgcctga tgagaggaca ctcacctttg ctgaaagatg gcaattatct 3060

tcacctgatg gagtagatac agatgatgat ttaccaaaat cgcgagcatc caaaagaacc 3120

tgtggtgtga atgatgatga aagtccaagc aaaattttta tggtgggaga atctccacaa 3180

gtgtcttcca gacttcagaa tttgagactg aataatttaa ttcccaggca acttttcaag 3240

cccaccgata atcaagaaac ttag 3264

SEQ ID NO：2

MARSSPYLRQ LQGPLLPPRD LVEEDDDYLN DDVEEDEESV FIDAEELCSG GVKAGSLPGC 60

LRVSICDENT QETCKVFGRF PITGAWWRVK VQVKPVVGSR SYQYQVQGFP SYFLQSDMSP 120

PNQKHICALF LKECEVSSDD VNKFLTWVKE VSNYKNLNFE NLRETLRTFH KETGRKDQKQ 180

PTQNGQEELF LDNEMSLPLE NTIPFRNVMT ALQFPKIMEF LPVLLPRHFK WIIGSGSKEM 240

LKEIEEILGT HPWKLGFSKI TYREWKLLRC EASWIAFCQC ESLLQLMTDL EKNALIMYSR 300

LKQICREDGH TYVEVNDLTL TLSNHMSFHA ASESLKFLKD IGVVTYEKSC VFPYDLYHAE 360

RAIAFSICDL MKKPPWHLCV DVEKVLASIH TTKPENSSDD ALNESKPDEV RLENPVDVVD 420

TQDNGDHIWT NGENEINAEI SEVQLDQDQV EVPLDRDQVA ALEMICSNPV TVISGKGGCG 480

KTTIVSRLFK HIEQLEEREV KKACEDFEQD QNASEEWITF TEQSQLEADK AIEVLLTAPT 540

GKAAGLLRQK TGLHAYTLCQ VNYSFYSWTQ TMMTTNKPWK FSSVRVLVVD EGSLVSVGIF 600

KSVLNLLCEH SKLSKLIILG DIRQLPSIEP GNLLKDLFET LKSRNCAIEL KTNHRAESQL 660

IVDNATRISR RQFPKFDAEL NISDNPTLPI SIQDKTFIFV RLPEEDAssQ SSKTNHHSCL 720

YSAVKTLLQE NNLQNAKTSQ FIAFRRQDCD LINDCCCKHY TGHLTKDHQS RLVFGIGDKI 780

CCTRNAYLSD LLPENISGSQ QNNDLDASSE DFSGTLPDFA KNKRDFESNV RLCNGEIFFI 840

TNDVTDVTFG KRRSLTINNM AGLEVTVDFK KLMKYCRIKH AWARTIHTFQ GSEEQTVVYV 900

VGKAGRQHWQ HVYTAVTRGR CRVYVIAEES QLRNAIMKNS FPRKTRLKHF LQSKLSSSGA 960

PPADFPSPRK SSGDSGGPST PSASPLPVVT DHAMTNDVTW SEASSPDERT LTFAERWQLS 1020

SPDGVDTDDD LPKSRASKRT CGVNDDESPS KIFMVGESPQ VSSRLQNLRL NNLIPRQLFK 1080

PTDNQET 1087

SEQ ID NO：3

ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc cctcctcacg gcgagcgctg 60

ccacgtcaga cgaagggcgc agcgagcgtc ctgatccttc cgcccggacg ctcaggacag 120

cggcccgctg ctcataagac tcggccttag aaccccagta tcagcagaag gacattttag 180

gacgggactt gggtgactct agggcactgg ttttctttcc agagagcgga acaggcgagg 240

aaaagtagtc ccttctcggc gattctgcgg agggatctcc gtggggcggt gaacgccgat 300

gattatataa ggacgcgccg ggtgtggcac agctagttcc gtcgcagccg ggatttgggt 360

cgcggttctt gtttgtggat cgctgtgatc gtcacttggt gagtagcggg ctgctgggct 420

ggccggggct ttcgtggccg ccgggccgct cggtgggacg gaagcgtgtg gagagaccgc 480

caagggctgt agtctgggtc cgcgagcaag gttgccctga actgggggtt ggggggagcg 540

cagcaaaatg gcggctgttc ccgagtcttg aatggaagac gcttgtgagg cgggctgtga 600

ggtcgttgaa acaaggtggg gggcatggtg ggcggcaaga acccaaggtc ttgaggcctt 660

cgctaatgcg ggaaagctct tattcgggtg agatgggctg gggcaccatc tggggaccct 720

gacgtgaagt ttgtcactga ctggagaact cggtttgtcg tctgttgcgg gggcggcagt 780

tatggcggtg ccgttgggca gtgcacccgt acctttggga gcgcgcgccc tcgtcgtgtc 840

gtgacgtcac ccgttctgtt ggcttataat gcagggtggg gccacctgcc ggtaggtgtg 900

cggtaggctt ttctccgtcg caggacgcag ggttcgggcc tagggtaggc tctcctgaat 960

cgacaggcgc cggacctctg gtgaggggag ggataagtga ggcgtcagtt tctttggtcg 1020

gttttatgta cctatcttct taagtagctg aagctccggt tttgaactat gcgctcgggg 1080

ttggcgagtg tgttttgtga agttttttag gcaccttttg aaatgtaatc atttgggtca 1140

atatgtaatt ttcagtgtta gactagtaaa ttgtccgcta aattctggcc gtttttggct 1200

tttttgttag acgaagcttg gtaccgagct cgatatcgcc accatggtga gcaagggcga 1260

ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca 1320

caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa 1380

gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac 1440

ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa 1500

gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa 1560

ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct 1620

gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta 1680

caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt 1740

caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa 1800

cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc 1860

cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac 1920

cgccgccggg atcactctcg gcatggacga gctgtacaag ggcaatggcg gcaatgctag 1980

cagcggcgca cctccagcag attttccgtc cccacggaag agctctggag acagtggagg 2040

acccagcaca ccgtcagcat ctccactccc tgtagtcaca gaccacgcca tgacaaatga 2100

tgtcacctgg agcgaggcct cttcgcctga tgagaggaca ctcacctttg ctgaaagatg 2160

gcaattatct tcacctgatg gagtagatac agatgatgat ttaccaaaat cgcgagcatc 2220

caaaagaacc tgtggtgtga atgatgatga aagtccaagc aaaattttta tggtgggaga 2280

atctccacaa gtgtcttcca gacttcagaa tttgagactg aataatttaa ttcccaggca 2340

acttttcaag cccaccgata atcaagaaac ttaggtcgac ccgggcggcc gcttcgagca 2400

gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa 2460

tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat 2520

aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggagatgtgg 2580

gaggtttttt aaagcaagta aaacctctac aaatgtggta aaatccgata aggatcgatc 2640

cgggctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc 2700

tgaatggcga atggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 2760

gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc 2820

ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt 2880

agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg 2940

ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac 3000

gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta 3060

ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat 3120

ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt cctgatgcgg 3180

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgcggatc tgcgcagcac 3240

catggcctga aataacctct gaaagaggaa cttggttagg taccttctga ggcggaaaga 3300

accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca 3360

gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct 3420

ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc 3480

ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg 3540

gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc 3600

agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctt gattcttctg 3660

acacaacagt ctcgaactta aggctagagc caccatgatt gaacaagatg gattgcacgc 3720

aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat 3780

cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt 3840

caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg 3900

gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag 3960

ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc 4020

tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc 4080

tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga 4140

agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga 4200

actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg 4260

cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg 4320

tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc 4380

tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc 4440

cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg 4500

gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgatggcc gcaataaaat 4560

atctttattt tcattacatc tgtgtgttgg ttttttgtgt gaatcgatag cgataaggat 4620

ccgcgtatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg 4680

acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta 4740

cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc 4800

gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat 4860

aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat 4920

ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata 4980

aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct 5040

tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa 5100

agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa 5160

cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt 5220

taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg 5280

tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca 5340

tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa 5400

cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt 5460

gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc 5520

cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa 5580

actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga 5640

ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc 5700

tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga 5760

tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga 5820

acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga 5880

ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat 5940

ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt 6000

ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct 6060

gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc 6120

ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc 6180

aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc 6240

gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc 6300

gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg 6360

aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata 6420

cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta 6480

tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc 6540

ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 6600

atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt 6660

cctggccttt tgctggcctt ttgctcacat ggctcgacag atct 6704

SEQ ID NO：4

ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc cctcctcacg gcgagcgctg 60

ccacgtcaga cgaagggcgc agcgagcgtc ctgatccttc cgcccggacg ctcaggacag 120

cggcccgctg ctcataagac tcggccttag aaccccagta tcagcagaag gacattttag 180

gacgggactt gggtgactct agggcactgg ttttctttcc agagagcgga acaggcgagg 240

aaaagtagtc ccttctcggc gattctgcgg agggatctcc gtggggcggt gaacgccgat 300

gattatataa ggacgcgccg ggtgtggcac agctagttcc gtcgcagccg ggatttgggt 360

cgcggttctt gtttgtggat cgctgtgatc gtcacttggt gagtagcggg ctgctgggct 420

ggccggggct ttcgtggccg ccgggccgct cggtgggacg gaagcgtgtg gagagaccgc 480

caagggctgt agtctgggtc cgcgagcaag gttgccctga actgggggtt ggggggagcg 540

cagcaaaatg gcggctgttc ccgagtcttg aatggaagac gcttgtgagg cgggctgtga 600

ggtcgttgaa acaaggtggg gggcatggtg ggcggcaaga acccaaggtc ttgaggcctt 660

cgctaatgcg ggaaagctct tattcgggtg agatgggctg gggcaccatc tggggaccct 720

gacgtgaagt ttgtcactga ctggagaact cggtttgtcg tctgttgcgg gggcggcagt 780

tatggcggtg ccgttgggca gtgcacccgt acctttggga gcgcgcgccc tcgtcgtgtc 840

gtgacgtcac ccgttctgtt ggcttataat gcagggtggg gccacctgcc ggtaggtgtg 900

cggtaggctt ttctccgtcg caggacgcag ggttcgggcc tagggtaggc tctcctgaat 960

cgacaggcgc cggacctctg gtgaggggag ggataagtga ggcgtcagtt tctttggtcg 1020

gttttatgta cctatcttct taagtagctg aagctccggt tttgaactat gcgctcgggg 1080

ttggcgagtg tgttttgtga agttttttag gcaccttttg aaatgtaatc atttgggtca 1140

atatgtaatt ttcagtgtta gactagtaaa ttgtccgcta aattctggcc gtttttggct 1200

tttttgttag acgaagcttg gtaccgagct cgatatcgct agcgctaccg gtcgccacca 1260

tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg 1320

gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg 1380

gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc 1440

tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc 1500

agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct 1560

tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg 1620

tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca 1680

agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg 1740

gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg 1800

accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact 1860

acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc 1920

tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtccg 1980

gactcagatc tcgagctcaa gcttccatgt cgtttacttt gaccaacaag aacgtgattt 2040

tcgttgccgg tctgggaggc attggtctgg acaccagcaa ggagctgctc aagcgcgatc 2100

ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg 2160

cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt 2220

cccaacagtt gcgcagcctg aatggcgaat ggcgctttgc ctggtttccg gcaccagaag 2280

cggtgccgga aagctggctg gagtgcgatc ttcctgaggc cgatactgtc gtcgtcccct 2340

caaactggca gatgcacggt tacgatgcgc ccatctacac caacgtgacc tatcccatta 2400

cggtcaatcc gccgtttgtt cccacggaga atccgacggg ttgttactcg ctcacattta 2460

atgttgatga aagctggcta caggaaggcc agacgcgaat tatttttgat ggcgttaact 2520

cggcgtttca tctgtggtgc aacgggcgct gggtcggtta cggccaggac agtcgtttgc 2580

cgtctgaatt tgacctgagc gcatttttac gcgccggaga aaaccgcctc gcggtgatgg 2640

tgctgcgttg gagtgacggc agttatctgg aagatcagga tatgtggcgg atgagcggca 2700

ttttccgtga cgtctcgttg ctgcataaac cgactacaca aatcagcgat ttccatgttg 2760

ccactcgctt taatgatgat ttcagccgcg ctgtactgga ggctgaagtt cagatgtgcg 2820

gcgagttgcg tgactaccta cgggtaacag tttctttatg gcagggtgaa acgcaggtcg 2880

ccagcggcac cgcgcctttc ggcggtgaaa ttatcgatga gcgtggtggt tatgccgatc 2940

gcgtcacact acgtctgaac gtcgaaaacc cgaaactgtg gagcgccgaa atcccgaatc 3000

tctatcgtgc ggtggttgaa ctgcacaccg ccgacggcac gctgattgaa gcagaagcct 3060

gcgatgtcgg tttccgcgag gtgcggattg aaaatggtct gctgctgctg aacggcaagc 3120

cgttgctgat tcgaggcgtt aaccgtcacg agcatcatcc tctgcatggt caggtcatgg 3180

atgagcagac gatggtgcag gatatcctgc tgatgaagca gaacaacttt aacgccgtgc 3240

gctgttcgca ttatccgaac catccgctgt ggtacacgct gtgcgaccgc tacggcctgt 3300

atgtggtgga tgaagccaat attgaaaccc acggcatggt gccaatgaat cgtctgaccg 3360

atgatccgcg ctggctaccg gcgatgagcg aacgcgtaac gcgaatggtg cagcgcgatc 3420

gtaatcaccc gagtgtgatc atctggtcgc tggggaatga atcaggccac ggcgctaatc 3480

acgacgcgct gtatcgctgg atcaaatctg tcgatccttc ccgcccggtg cagtatgaag 3540

gcggcggagc cgacaccacg gccaccgata ttatttgccc gatgtacgcg cgcgtggatg 3600

aagaccagcc cttcccggct gtgccgaaat ggtccatcaa aaaatggctt tcgctacctg 3660

gagagacgcg cccgctgatc ctttgcgaat acgcccacgc gatgggtaac agtcttggcg 3720

gtttcgctaa atactggcag gcgtttcgtc agtatccccg tttacagggc ggcttcgtct 3780

gggactgggt ggatcagtcg ctgattaaat atgatgaaaa cggcaacccg tggtcggctt 3840

acggcggtga ttttggcgat acgccgaacg atcgccagtt ctgtatgaac ggtctggtct 3900

ttgccgaccg cacgccgcat ccagcgctga cggaagcaaa acaccagcag cagtttttcc 3960

agttccgttt atccgggcaa accatcgaag tgaccagcga atacctgttc cgtcatagcg 4020

ataacgagct cctgcactgg atggtggcgc tggatggtaa gccgctggca agcggtgaag 4080

tgcctctgga tgtcgctcca caaggtaaac agttgattga actgcctgaa ctaccgcagc 4140

cggagagcgc cgggcaactc tggctcacag tacgcgtagt gcaaccgaac gcgaccgcat 4200

ggtcagaagc cgggcacatc agcgcctggc agcagtggcg tctggcggaa aacctcagtg 4260

tgacgctccc cgccgcgtcc cacgccatcc cgcatctgac caccagcgaa atggattttt 4320

gcatcgagct gggtaataag cgttggcaat ttaaccgcca gtcaggcttt ctttcacaga 4380

tgtggattgg cgataaaaaa caactgctga cgccgctgcg cgatcagttc acccgtgcac 4440

cgctggataa cgacattggc gtaagtgaag cgacccgcat tgaccctaac gcctgggtcg 4500

aacgctggaa ggcggcgggc cattaccagg ccgaagcagc gttgttgcag tgcacggcag 4560

atacacttgc tgatgcggtg ctgattacga ccgctcacgc gtggcagcat caggggaaaa 4620

ccttatttat cagccggaaa acctaccgga ttgatggtag tggtcaaatg gcgattaccg 4680

ttgatgttga agtggcgagc gatacaccgc atccggcgcg gattggcctg aactgccagc 4740

tggcgcaggt agcagagcgg gtaaactggc tcggattagg gccgcaagaa aactatcccg 4800

accgccttac tgccgcctgt tttgaccgct gggatctgcc attgtcagac atgtataccc 4860

cgtacgtctt cccgagcgaa aacggtctgc gctgcgggac gcgcgaattg aattatggcc 4920

cacaccagtg gcgcggcgac ttccagttca acatcagccg ctacagtcaa cagcaactga 4980

tggaaaccag ccatcgccat ttgctgcacg gggaagaagg cacatggctg aatatcgacg 5040

gtttccatat ggggattggt ggcgacgact cctggagccc gtcagtatcg gcggaattac 5100

agctgagatc tagcggcgca cctccagcag attttccgtc cccacggaag agctctggag 5160

acagtggagg acccagcaca ccgtcagcat ctccactccc tgtagtcaca gaccacgcca 5220

tgacaaatga tgtcacctgg agcgaggcct cttcgcctga tgagaggaca ctcacctttg 5280

ctgaaagatg gcaattatct tcacctgatg gagtagatac agatgatgat ttaccaaaat 5340

cgcgagcatc caaaagaacc tgtggtgtga atgatgatga aagtccaagc aaaattttta 5400

tggtgggaga atctccacaa gtgtcttcca gacttcagaa tttgagactg aataatttaa 5460

ttcccaggca acttttcaag cccaccgata atcaagaaac ttagttttat ttcaaattgt 5520

tccgagtaac tatgtttttc tattggagac aaaatgaaca tcgtaacgtc aaagtaccaa 5580

gataagaatt ctgcagtcga cggtaccgcg ggcccgggcg gccgcttcga gcagacatga 5640

taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta 5700

tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag 5760

ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt 5820

tttaaagcaa gtaaaacctc tacaaatgtg gtaaaatccg ataaggatcg atccgggctg 5880

gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg 5940

cgaatggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 6000

gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 6060

ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 6120

cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt 6180

agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 6240

aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt 6300

gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 6360

aaatttaacg cgaattttaa caaaatatta acgcttacaa tttcctgatg cggtattttc 6420

tccttacgca tctgtgcggt atttcacacc gcatacgcgg atctgcgcag caccatggcc 6480

tgaaataacc tctgaaagag gaacttggtt aggtaccttc tgaggcggaa agaaccagct 6540

gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat 6600

gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc 6660

aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac 6720

tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact 6780

aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta 6840

gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag cttgattctt ctgacacaac 6900

agtctcgaac ttaaggctag agccaccatg attgaacaag atggattgca cgcaggttct 6960

ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc 7020

tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc 7080

gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc 7140

acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg 7200

ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag 7260

aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc 7320

ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt 7380

cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc 7440

gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc 7500

tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg 7560

ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag 7620

cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg 7680

cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg 7740

aaatgaccga ccaagcgacg cccaacctgc catcacgatg gccgcaataa aatatcttta 7800

ttttcattac atctgtgtgt tggttttttg tgtgaatcga tagcgataag gatccgcgta 7860

tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg 7920

ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa 7980

gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc 8040

gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg 8100

gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 8160

tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 8220

caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc 8280

ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 8340

gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt 8400

aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 8460

ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 8520

atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 8580

gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 8640

gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac 8700

atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 8760

aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 8820

actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 8880

aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 8940

tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 9000

ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 9060

agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 9120

tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 9180

aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 9240

gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 9300

atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 9360

gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 9420

gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 9480

tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 9540

accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 9600

ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 9660

cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 9720

agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 9780

ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 9840

tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 9900

ttttgctggc cttttgctca catggctcga cagatct 9937

SEQ ID NO：5

MVSKGEELFT GVVPILVELD GDVNGHKFSV SGEGEGDATY GKLTLKFICT TGKLPVPWPT 60

LVTTLTYGVQ CFSRYPDHMK QHDFFKSAMP EGYVQERTIF FKDDGNYKTR AEVKFEGDTL 120

VNRIELKGID FKEDGNILGH KLEYNYNSHN VYIMADKQKN GIKVNFKIRH NIEDGSVQLA 180

DHYQQNTPIG DGPVLLPDNH YLSTQSALSK DPNEKRDHMV LLEFVTAAGI TLGMDELYKG 240

NGGNASSGAP PADFPSPRKS SGDSGGPSTP SASPLPVVTD HAMTNDVTWS EASSPDERTL 300

TFAERWQLSS PDGVDTDDDL PKSRASKRTC GVNDDESPSK IFMVGESPQV SSRLQNLRLN 360

NLIPRQLFKP TDNQET 376

SEQ ID NO：6

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggc 720

aatggcggca atgctagcag cggcgcacct ccagcagatt ttccgtcccc acggaagagc 780

tctggagaca gtggaggacc cagcacaccg tcagcatctc cactccctgt agtcacagac 840

cacgccatga caaatgatgt cacctggagc gaggcctctt cgcctgatga gaggacactc 900

acctttgctg aaagatggca attatcttca cctgatggag tagatacaga tgatgattta 960

ccaaaatcgc gagcatccaa aagaacctgt ggtgtgaatg atgatgaaag tccaagcaaa 1020

atttttatgg tgggagaatc tccacaagtg tcttccagac ttcagaattt gagactgaat 1080

aatttaattc ccaggcaact tttcaagccc accgataatc aagaaact 1128

Claims (40)

1. polypeptide construct, it comprises detectable viable cell reporter molecule, this molecule is by having less than 112, the group of 000 Dalton molecular weight is connected at least one cell cycle phase dependency positioning controling element, being positioned at G1 and changing during the S phase of described element, the transposition of wherein said construct in mammalian cell shows the cell cycle position.

2. according to the polypeptide construct of claim 1, wherein said group has less than 100,000 daltonian molecular weight.

3. according to the polypeptide construct of claim 1, wherein said group has less than 50,000 daltonian molecular weight.

4. according to the polypeptide construct of claim 1, wherein said group has less than 25,000 daltonian molecular weight.

5. according to the polypeptide construct of claim 1, wherein said group has less than 10,000 daltonian molecular weight.

6. according to the polypeptide construct of claim 1, wherein said group has less than 1,000 daltonian molecular weight.

7. according to the polypeptide construct of claim 1, wherein said group has less than 700 daltonian molecular weight.

8. according to the polypeptide construct of claim 1, wherein said group has less than 500 daltonian molecular weight.

9. according to the polypeptide construct of claim 1, wherein group is a polypeptide.

10. according to the polypeptide construct of claim 9, wherein said peptide group is seven peptides.

11. according to the polypeptide construct of claim 10, wherein said seven peptides are glycine-l-asparagine-glycine-glycine-l-asparagine-L-Ala-Serine (GNGGNAS).

12. according to each polypeptide construct of aforementioned claim, wherein cell cycle phase specificity dependency positioning controling element is selected from the peptide group that Rag2, Chaf1B, Fen1, PPP1 R2, helicase B, sgk, CDC6 or its motif are formed, the phosphorylation dependency Subcellular Localization structural domain (PSLD) in the C-terminal spatial control district of described motif such as helicase B.

13. according to each polypeptide construct of claim 1 to 12, wherein the viable cell reporter molecule is selected from fluorescin, enzyme reporter molecule and antigenicity sign.

14. according to the polypeptide construct of claim 13, wherein said fluorescin is selected from green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Emerald and J-Red.

15. according to the polypeptide construct of claim 13, wherein said enzyme reporter molecule is halo-tag (Promega).

16. according to the polypeptide construct of aforementioned each claim, wherein cell cycle phase dependency positioning controling element is PSLD.

17. according to the polypeptide construct of aforementioned each claim, wherein reporter molecule is that EGFP and cell cycle phase dependency positioning controling element are PSLD.

18. polypeptide construct comprises the aminoacid sequence of SEQ ID No.5.

19. coding is according to the nucleic acid construct of any polypeptide construct of aforementioned each claim.

20. additionally comprising and be operably connected to and be controlled by at least one cell cycle dependent/non-dependent, the nucleic acid construct of claim 19, wherein said construct express controlling elements.

21. the nucleic acid construct of claim 20, wherein said expression controlling elements are ubiquitin C promotor or CMV promotor.

22. according to each nucleic acid construct of claim 19 to 21, it comprises the sequence of CMV promotor and coding PSLD and EGFP or J-Red.

23. according to each nucleic acid construct of claim 19 to 21, it comprises the sequence of ubiquitin C promotor and coding PSLD and EGFP or J-Red.

24. comprise each the carrier of any nucleic acid construct of claim 19 to 23.

25. according to the carrier of claim 24, wherein said carrier is virus vector or plasmid.

26. according to the carrier of claim 25, wherein said virus vector is adenovirus carrier or lentiviral vectors.

27. use according to each the host cell of nucleic acid construct transfection of claim 19 to 23.

28. according to the host cell of claim 27, wherein said cell is people's cell.

29. comprise stable cell lines according to one or more host cells of one of claim 27 or 28.

30. be used for determining the purposes of the cell cycle position of mammalian cell according to each polypeptide construct of claim 1 to 18.

31. be used for the method for the cell cycle position of definite mammalian cell, described method comprises:

A) in cell, express according to each nucleic acid construct of claim 19 to 23; And

B) signal of launching by the monitoring reporter molecule is determined the cell cycle position.

32. determine the method for test agent to the influence of the cell cycle position of mammalian cell, described method comprises:

A) lacking and existing under the situation of described test agent, in described cell, expressing according to each nucleic acid construct of claim 19 to 23; And

B) determine the cell cycle position by the signal launched of monitoring reporter molecule, wherein the difference between measured the transmitting has been indicated the influence of the cell cycle position of test agent pair cell when lacking and have described test agent.

33. determine the method for test agent to the influence of the cell cycle position of mammalian cell, described method comprises:

A) exist under the situation of described test agent, in described cell, expressing according to each nucleic acid reporter constructs of claim 19 to 23; And

B) signal of launching by the monitoring reporter molecule is determined the cell cycle position,

The given value that the signal of being launched when c) relatively having test agent is transmitted when lacking test agent;

Wherein when having test agent measured transmit and the given value when lacking test agent between difference indicated the influence of the cell cycle position of test agent pair cell.

34. determine the method for test agent to the influence of the cell cycle position of mammalian cell, described method comprises:

A) provide and contain each the cell of nucleic acid construct of with good grounds claim 19 to 23;

B) respectively at first and second cell masses that have and lack test agent and allow to cultivate under the condition of express nucleic acid reporter constructs described cell; And

C) measure the signal that the reporter molecule in described first and second cell masses is launched;

Difference in wherein said first and second cell masses between measured the transmitting has been indicated the influence of described test agent to the cell cycle position of described cell.

35. determine the method for the influence of mammalian cell cycle pair cell process, described process can first can detect reporter molecule and measures in what test agent changed by known response, described method comprises:

A) exist under the situation of described test agent, in described cell, expressing according to each the second nucleic acid reporter constructs of claim 19 to 23;

B) determine the cell cycle position by monitoring the signal that second reporter molecule launches; And

C) monitoring described first can detect the signal that reporter molecule is launched,

Wherein the determined cell cycle position of step b) and first relation that can detect between the signal that reporter molecule launches has indicated whether described cell processes is the cell cycle dependency.

36. be used for measuring the active purposes of CDK2 of cell according to each polypeptide construct of claim 1 to 18.

37. the active method of CDK2 said method comprising the steps of in the mensuration cell

A) in cell, express according to each nucleic acid construct of claim 19 to 23, and

B) signal of launching by the monitoring reporter molecule is determined the CDK2 activity.

38. be used for determining the method for test agent to the active influence of CDK2 of mammalian cell, described method comprises:

A) lacking and existing under the situation of described test agent, in described cell, expressing according to each nucleic acid construct of claim 19 to 23; And

B) determine the CDK2 activity by the signal launched of monitoring reporter molecule, wherein the difference between measured the transmitting has indicated test agent to the active influence of CDK2 in shortage and when having described test agent.

39. determine the method for test agent to the active influence of mammalian cell CDK2, described method comprises:

A) exist under the situation of described test agent, in described cell, expressing according to each nucleic acid construct of claim 19 to 23; And

B) determine the cell cycle position by the signal of monitoring reporter molecule emission,

The given value that the signal of being launched when c) relatively having test agent is transmitted when lacking test agent;

When wherein having test agent measured transmit and the described given value when lacking test agent between difference indicated the active influence of test agent pair cell CDK2.

40. according to each method of claim 37 to 39, wherein said test agent is the form or the chemical entities of electromagnetic radiation.

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