Gris1 in the leukemia that Graffi rat the section is inside a new usually integrate the leukemia that position respond virus:Be sliced by 於 of first
Laboratoire de Biologie 摩尔 é culaire, D é partement des science Biologiques, Universit é du Qu é bec à Montr é al, Qu é bec, Canada,1 The microbiology, allergy of the laboratory numerator with there is catching academic association of nation disease, Bethesda, Maryland2'k, http://www.100md.com
Received the 2002 July 3/ the 2002 of the general acknowledgement September 16'k, http://www.100md.com
68:540-558,1957). Gris1 ( integrate the position because of the Graffi 1). 滤 is over the sex poison the 菌 's integration atGris1 Disheveled hair now 13% of tumor analysis. ThatGris1 The place was shot there end the department the district for of rat chromosome 6 by 映 ,85 kbs heading up to swim that cyclin D2 the gene. Gris1 Major cyclin that cause 6.5 kbs of that normals of overexpressions report card that report card that D2 is besides too outside lure that new expression, two 择 a the ground join together 1.1 kbs the cyclin D2 gene that change from that to the secret code everything the head to carry 17 kDa of cyclin D2. The report card's expression of that this 1.1 kbs is a particular tumor to ～Gris1'k, http://www.100md.com
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Graffi complicated 滤 is over the sex poison the 菌 is the first from have no the cell insulation 滤 liquid Ehrlich tumor with almost exclusive respond the myeloid Leukemia when in the infant that rat that some strains inoculate (10). Is from that 滤 over the special features that sex poison the 菌 's synthesis insulation and(30). The replication right and wrong of these two numerators often resembleses in the construction, and apart from Make duplicate the GV-1.2 to lure a short delitescence of disease with ～ Period with in the U3 performance in the enhancer the duplicate of 60 bps of a perfections District that hope earnestlies computer terminal repetition(( LTR)30). New-born BALB/ c And the NFS rat inoculate with one of the replication of two numeratorses Or because that parental admixture develop, to different degree, The hepatosplenomegaly, thymic extended the language, generalization the lymphadenopathy, And present to public in the inside's anemic leukemic.So that, Graffi rat section animal The virus( MuLV) of right research leukemia were considered a good model Myeloid mechanism leukemia inductive method with go forward And arrive to recognize the clear and cloak-and-dagger proto- oncogenes.
The Graffi virus lures the leukemia's accurate mechanism Is not understanding that like. However, making of the cell's proto- oncogenes activity Quilt insertional the mutagenesis is a main step in the leukemic procedure. We in times before have to report the making of c the activity-myc,Fli1,Pim1, And Spi1/PU.1Integration of Graffi virus gene at 20,10,3.3, And 3.3%, respectively, tumor test(6). The in order to identifies the new cell to proto- oncogenes, we contain replication with Meant an unusual 滤 that come from the Graffi over the integration position that sex poison 菌 that the special features respond the MuLV Tumor.6w-3&, http://www.100md.com
In this research, we report confirm with describe Gris1( because the Graffi integration make 1 locate), a new usually position Respond the MuLV integration in the tumor in the Graffi. 滤 is over the sex poison the 菌 's integration AtGris1 Gris1 The place was shot the rat chromosome by 映 6 of the district of the end department And were located to face the upper stream cyclin D2 the gene of 85 kbp. That 滤 is over the sex poison 菌 of Integrate atGris1 Cyclin main report card D2 gene and incitement expression New, still- Uncharacterized alternative plan the report card of 1.1 kbs From the homology's gene.
Main of tumor. NFS/ N is with the BALB/ c the rat were purchased from Frederick Cancer to study the equipments, Frederick, Md., with from check the 尔斯 river the company, St. Constant, Quebec, Canada, respectively. The freshman's rat(<48H past) about use what～ inoculate the intraperitoneally105 PFU at 0.1 to 0.2 毫s rose the percolation 滤 the sex poison the 菌 's 浮 at superficial.k63{ie, 百拇医药
Genomic- DNA with- RNA analysis. High mark son- The weight genomicDNA were excerpted from the congelation Normal is with the thin 纱织 of leukemic 品 quilt according to the standard procedure that describe In times before(31). µGenomic g Come from what tumor of the DNA or control organ is digested because of an adequacy Limit the endonuclease, and separate on first 0.7% the agarose gel, with Transfer a nylon thin film.( Osmonics)k63{ie, 百拇医药
Total amount RNA is from the frozen normal with the thin 纱织 of leukemic the 品 were excerpted Because of the TRIzol reagent( life technique) according to that The instruction makes the operator. Analyze to 於 north,20 µThe g of the total RNA at 1.2% A 荃 agarose on were separated According to the gel that describe in times before(34) and transfer the nylon Thin film.( Osmonics)
All stain at 50% formamide-10% the 士 get the 郎 the sulfate-0.5% the inside is made the 杂 to hand over 钠 dodecyl sulfate( SDS)-1 the M NaCl is then 42 ° C because that is appropriate Fish for. By arbitrarily of the handbook extension perform the lifelike usage oligohexamers( Pharmacia P- L biochemistry of, Montreal, Canada). The thin film were washed At 2x x SSC is 0.15 sour salt of 檬 of M NaCl-0.015 M 钠柠 ) for 30 The least is then 42 ° C with at 0.2x^!\?, http://www.100md.com
滤 is over the sex poison the inserting of 菌 make enlarge to locate. Enlarge that provirus- The cell's DNA 联 connects to be circulated By the PCR(37). Usage of extending specific medicine of a kind of biotinylated handbook is that Graffi U3 district that LTR(5' AGGCGCAAGGTCGTTTCAGGTCTTTGGG 3') with 1 5 partially deteriorated drive handbook that describe( FP) In times before(36). In the streptavidin glazed on magnetism grain son.(Boehringer Mannheim) The second PCR is then in the recovered biotinylated on circulate Amplicon usage a 筑 nest Graffi virus- Specific medicine handbook(5' ATGGGTCTCTTGAAACTGCTGAGGG 3') with on matching the nondegenerated handbook allotment FP handbook is in the first PCR convenient(5' CAGTTCAAGCTTGTCCAGGAATTC 3'). Because of the tumor 6 with 8, at the 滤 in the district over the sex poison the 菌 's integration at Gris1 AndCcnd2 The specific medicine is that genomic district atGris1 AndCcnd2 ( a The handbook is each 10 kbp) in the combination one of convenient two handbookses The specific medicine is a Graffi LTR. The handbook specific medicine is a Graffi The LTR is a Graffi 254-229(5' GGGGCAACCTGGAAACATCTGATGGG 3') with Graffi 10-34.(5'CCCCACCATAAGGCTTAGCAAGCTAG 3') genomic The specific medicine handbook designs the winnings according to the preface row In the Ensembl rat the genome wait upon the 器 To 於 chromosome 6 district 128.05 to 128.15 Mb. Handbook Is according to following each item: GRIS1F,5'GTCTCTTTTGTTGCTTTGAGGGGC 3';GRIS2F, 5'GAGAAGGTGAATGGTTTTTATGCCC 3';GRIS3F,5'CAGAGTTAGAACCTGGGTGTGTGC 3'; GRIS4F,5' CTGACAAGCCGCCCTAGGTG 3'; GRIS5F,5' GTCCATTTTCCAGGCAGGACTGAG 3'; GRIS6F,5' CTGAGCCCTCCATATCAGTCATCA 3'; GRIS7F,5' TTGATTTTTGCCTGCTTGTCCTCAC 3'; GRIS8F,5' TCTTCCACTTCCTGGTTTGGTATC 3'; GRIS9F,5'CAGGAGTAACGGGAGAGAGCTA 3'; with the GRIS10F,5' TGCGTTAAGAGGACTGAGACCC 3'.
The 映 that inherit the gene shoots. Gris1The southern stain 映 of quilt shoots two combinations's analysises inherit the gene of The cross: ( NFS/ N or C58/ Jx The 12 musculus of the 12 Greek alphabet of Greek alphabet)F1 x M. (20) and( NFS/ Nx The 12 spretus of Greek alphabet)x M. spretus Or C58/ J(1). Descendant These the crosses have to be typed because exceed 1,200 person's allotments that make the marks Exceed to have of 19 autosomes with X chromosome. Combine the distance Decide the recombinants from the arithmetic figure quilt, and loci is By reduce the minimal order the arithmetic figure to reunite.|]?r), http://www.100md.com
Genomic- The replication isolates.The BAC genomic make duplicates to include thatGris1 By the reviewing RPCI-129 S6 part 2 rat BAC library( BACPAC Resources; CHORI, California Auckland.) Because ofGris1|]?r), http://www.100md.com
Case shot hunting rifle preface row.Case shot hunting rifle the library quilt construct to consider as to in times before describe (43). Brief, the BAC DNA is a large-scale alkaline preparation of quilt The symptoms that there is the big reduces the 退 structure the material. (Qiagen) µg) Does the sonicated is a nuclease to fix the buffer inside in the mung bean son(30 毫莫 ear 钠 Acetate,50 毫莫 ear NaCl,1 毫莫 ear ZnCl2,5%glycerol). Fragment be over Then 30 ° Cs are to have 40 U of 30 leasts is fixed the mung bean the son nuclease.(Amersham- The Pharmacia livings the 化 technology) the sample is an electrophoresed At 1% agarose on gum 化 , with 800- Fragment for 1,200 bps, is a gum 化 The 洁 is clean to enter that with ligated insideEcoThe recreational vehicle make duplicates the position pBlueScript 2 KS(+).( Stratagene) the tubal ligation product is a conversion Enter to use the Dh5 of the chemistry ability inside{alpha} The alkali symptoms that DNA replication were prepared was reduced the 退 by small scale because of The Miniprep equips from the Qiagens. That preface the row respond is Use big- 颜料 terminator the period circulate the preface row the material the edition 2.0( applied Biosystems, California city that keep and raise.). Respond to be involved a model number 3700 DNA analysis.( applied Biosystems)
Game. The cDNA ends( game) to extend quick 5' with 3' experiment Were circulated to use the GeneRacer material. ( Invitrogen) that game- prepare the good appearance The cDNA pond were acquired the usage the GeneRacer 寡 the 醣 ( dT) handbook On the tumor, total RNA of F6. 5 Of enlarge' end to express Preface row the label( ET) AW060277 is a finished usage that GeneRacer 5' handbook with ET- Specific medicine handbook 5' GTCTTGGTTAGTGTGGCGGCCTTA 3'. GeneRacer 3' handbook with that ET- Specific medicine handbook 5' TCACACTAAGGCCGCCACACACTAA 3' the habit 於 acquire 3' end.-yallkp, http://www.100md.com
The cell cultivates the circumstance, plasmid building with the transfections.NIH 3 Dulbecco's of T3 cells at correction eagles were supported The medium( life technique) uses 10% calf 浆 liquid complement ( life technique). pCMV-The D2trc constructs the quilt the replications to do The beguilement quilt that that is complete acquires of is sliced the head the cyclin D2 preface row that carry By the PCR because of following and various handbooks: 5'TTTCACCCTCTAGAATTTCCATG 3' with 5' AGGAGGTAAGGGAAGCTTTCC 3'. The fragment is a replication At thatHindIII-XbaThe position wireless of my pRcCMV inducement. (Invitrogen) The Transfections uses to be done the Polyfect( Qiagen) according to The instruction of the manufacturing operator.
The antibody preparation, protein draw outs with the western person's stain. The last 20 kind that come from the C- computer terminal of protein and objection contain the ammonia 基 sour The be over produces. Brief, right should of cDNA preface row Is a PCR to use the glutathione in the physical stature to extend and make duplicate S- The tranferase enters pGEX-4 Ts-1ses the protein in the vector.(Amersham Biosciences) After inductive method, bacterially squeeze out the thing to is delivered the glutathione- Sepharose 4 protein 洁 that B column( Amersham Biosciences), with were made clean Is a skin bottom of into New Zealand white rabbit inside inject. The 浆 liquid collected immediately after of 凝 piece the 缩 enter and store at -70 ° C. NIH 3 T3s contain pCMV- D2trc fibroblasts transfected The plasmid were washed to twice there is ice in the RIPA- A cold PBS is with the lysed Buffer(0.15 M NaCl,1% NP-40,0.5% 钠 deoxycholate,0.1% SDS,0.05 M Tris- HCl[ pH 8]) with complete the protease depressant Chicken tail wine( the Roche applied science) for 30 then 4 ° C. The protein is minimum Come from the squeezing of tumor out the thing with the thin 纱织 the 品 quilt prepare in the SDS-The urea buffer is inside (0.5% SDS,8 M urea). The protein's concentration at that is different Squeeze outing thing to is measured to use to living the 化 Rad the protein to analyze. Opponent Lysate protein quantity(30 to 80µg) Dilute At 2x The 抽 takes the sample buffer(30% glycerol,4% SDS,160 毫莫 ear Tris- HCl[pH 6.8],10%ß- mercaptoethanol, with 0.02% bromophenol 蓝色 of) with boil the 沸 to 3 the 最 ? the The protein is separately at 12% on SDS- Polyacrylamide gum 化 electricity the 泳 move with the electrophoretically Turn to move to the Immobilon- P polyvinylidenedifluoride thin film (Millipore). The thin film were jamed to include in the PBS 0.2% Tween 20 with 4% 浮渣 milk powder( PBS- Tween- milk) for 1 h at Room 温 . Incubation because that make the versa 洁 clean slice the head to carry Sour antibody of ammonia of cyclin D2 缩 (1:100 in the dilution in the PBS- Tween- milk) Or the anti slice the head to carry the cyclin D2 浆 the liquid( in the PBS- Tween- milk of 1:5,000 dilution) Is for 1 h is in the room 温 movement. In the incubation's 後 , The stain includes in the PBSs 0.2% Tween 20 insides is washed( PBS- Tween) And embrace for have the versa rabbit immunoglobulin G the peroxidase of 1 h Combination.(1:5,000 in the PBS- Tween- milk)( BD Pharmingen) that is immune Of the synthesis were shown the usages the ECLs plus chemiluminescence Reagent.( AmershamBiosciences)
ConfirmGris1, in the position that the integration in the Graffi is a to usually respond the MuLV tumor.Identify the new proto that stimulate of quilt- The oncogenes responds the MuLV tumor in the Graffis, drive Sorensen PCR method that describe is with the colleague(37)37Were applied the 37 於 the winning the 格状 to automate the DNA to attackstone the on the side 5' come from the tumor 12 of Graffi proviruses be over. A the special genomicPCR fish for 400 bps is produced and used to investigate from that tumor 30 of other tumor DNA of patch. On the southern stain, this fish for( fish for a[ diagram1B]) can quilt is discovered then the alignment's allele is in the tumor 12 DNA inside and at an other tumor( tumor 4) of inside( diagram1A).More the low quilt is again normal allele of the alignment's allele mightiness and ～相 is in the tumor 4 suggest that 滤ed the sex poison the 菌 's integration at this position for that tumor partially is a clonal.Two proviruseses both are all at the homology in that place of settle the directions aspect and is located besides each other outside of 1 kbp. Gris1.kn, http://www.100md.com
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Figure 1. Southern stain analysis The Graffi responds the MuLV 的白血 ? the ( a) normal spleen is with the tumor DNA Digest because ofCheat嗨 with were fish fored on make the 杂 hand overing. (B) Limit the map thatGris1 Place. Position with Settling of proviruses directions quilt arrows designation.映 that that fish for and used to discover then arranging the language and because the heredity gene shoot isManifestation is such as the solid box. ( a with the B, respectively) fish for an is The quilt PCR acquires, and fish for the B origins 於 genomic DNALambda the replication. The abbreviation is to limit the endonucleases: E,EcoRI; B,Cheat嗨 ; R,EcoRecreational vehicle ; S,SacI; P,PstI; X,XbaI. ThatXbaMy fragment is a length 13 kbp.
Gris1 Fish for on were acquired by PCRing to use to investigate the genomicLambda to come from the rat the phage library of the 129/ Sv the winning the replication to express that germs the lineGris1 Place. The replication of a 8 kbses is a recovereds with use to construct a restrict map of amenity with get from a long afterwards is used special to fish for( fish for the B) to shoot for 映 of inheritting the gene( diagram1B). 1B:xcf#ch, 百拇医药
The 映 that inherit the gene shootsGris1.Decide that the heredity the gene- Map positionGris1, we investigate Inherit the cross in heredity gene of two combinations is inside of this make the mark's person. Combine the inside the cross, southern stain the analysis to identify in the first 6.8 Kb ofEcoRI fragment atM. M. musculus And the fragment of 5.2 kbs In the NFS/ N rat.Combine the inside in the cross's the second,Sac2 Digest Produce the fragment of 3.0 kbs atM. spretus And the fragment of 1.2 kbs In the NFS/ N rat. The style of the different fragment heredity At those two the crosses's combination quilt and ～相 than 1,200 of The person who make the mark in times before typeses and the 映 shoot. The linkage is observed because of Make the mark's chromosome of person at on end department 6. Gene order of sequence is with the recombinational The distance is according to following each item: Raf1 (3.0 ± 1.1)-Tnfr1 Ltbr (0.4 ± 0.4)-Scnn1a Gris1 Ccnd2 (5.2 ± 1.5)-Kras2.
Quilt 滤 is over the sex poison the gene that the 菌 's integration stimulate confirm atGris1. Or by the 滤 over the sex poison the 菌 's integration bluntness 化 atGris1 That 8 kbs ofGris1 The genomic Lambda is the expression's preface row replication. However, have no a transcriptional unit can quilt Northerner detection Of stain analysis.( the data does not show) generally and widely behaves to know the gene to make the activity Integration that quilt 滤 is over the sex poison the 菌 exceed the big distance can take place(12,21, 35).The so that, we searches the candidate gene at a district About and about 200 kbp environments thatGris1Insert to locate. Contig thatGris1 The district were acquired a rat by the reviewing 129/ Sv genomic BAC library. Two hand over the 叠 to handle the BAC replication is Recovered and used to produce the district's a restrict map ( diagram2). The most long replication recovered is in the sample case shot the hunting rifle inside were used The preface row designs to acquire the preface row of a 2.5 fold layerses the surplus 200 District for kbpGris1. The each preface row then were analyzed Personally oppose the NR, decibel with the HTGS 核甘 database Use the fresh breeze 运 the calculate way then. We preface row is discoversed same alike at that Database because of two already gene that know and because some ETs are irrelevant Arrive already gene that know.Those two already gene that know is for the triose phosphate is those isomerase(Tpi) and cyclin D2(Ccnd2). Position that Comparison of two genes arriveGris1 ( diagram2). TpiThe gene heading ups to swim to about is placed 30 kb Gris1, however thatCcnd2The gene about were placed 85 kbp Downstream ground( diagram2).Decide whether to express that or notTpi And Ccnd2 Gene quilt 滤 is over the sex poison the 菌 's integration changes atGris1, Similar to anyly present of genomic fragmentTpi OrCcnd2 Were used in the northern stain analysis. Even we Place thatGris1 The place has 30 kbp fromTpi, did not change In the report card's expression description of the gene's 1.5 kbses Can be a convenient 滤 of tumor in integration hair over the sex poison 菌 nowGris1 Comparison because of the other Graffi tumor with control the organ( the data is not Show). Two tumorGris1 Were arranges agained( tumor 4 with 12) Emerge in the anticipant size normal cyclin D2 information 6.5 Kb, but amazingly of, two tumors present an affixture of The band of the mightiness's 1.1 kbses( diagram3A). This new cyclin D2- Specific medicine band Is in the tumor absentGris1 Have no quilt to arrange again( tumor 7,10, with 11). Tumor 6 with 8 also present a mightiness of 1.1 kb of Band, even our must not detection the DNA arrange the language again at ThatGris1There is place that fish for an and B. Later on locate from the integration Were located about 85 kbps keep off the cyclin D2 the gene, we the deep thought That tumor 6 with 8 most and probably sheltered a 滤 the sex poison the 菌 's integration It is some to be placed inGris1 Fish for a with the boundary that the B discovers. Have already take placed at thatGris1 Extended the 6 different from 8 because of the handbook of doubles tumor, a Specific medicine is because of the Graffi MuLV U3 LTR and another origins 於 Each 10 of kbps at cyclin D2 gene with thatGris1 Place. This procedure also is circulated because of the U3 and genomic handbook right Match the another retroviral to settle the directions. PCR product Use a kind of U3 LTR- The specific medicine were made the 杂 to hand over to fish for. According to expect, we Can a special U3 is discovered- The specific medicine fragment is each tumor because of Combination for there is handbook( the result not show)ing, certain Provirus emergence at thatGris1 Place is in the tumor 6 with 8 Then 4 with 5.5 kbp, respectively, heading up to swim thatXbaMy position 5 Is which' the detection boundary of the first analysis. That Provirus emergence at thatGris1 In the tumor 6 insides's place with 8 is Also analyze the proof the usage by the southern stain three different Get from the genomic to fish for. ( consequently not show) consider as the decision quilt Proviruses PCR, that come from the tumor 6 similar to 8 is atly settle the directions aspect Consider as those in the tumor 4 with 12 inside.So that, outlying 4 tumorses 30(13%) Present a the Graffi MuLV integrate at thatGris1Place is with the expression A the cyclin D2 of new 1.1 kbses- Specific medicine mRNA.
fig.ommitteed/m0l49, 百拇医药
Figure 2. Predicted the actual map ThatGris1 Genomic district. The BAC uses the ～ replication the recovered thatGris1 MluI-,ClaI-, withPvuI- Digest the BAC replication. On the map of the small arrows instructions that 滤 is over the sex poison the 菌 's integration position with settle the directions to tumor 6 With 8 with the tumor 4 with 12. ThatGris1 Use of fish for is Show and have no quilt painting according to the comparison to decide./m0l49, 百拇医药
fig.ommitteed/m0l49, 百拇医药
Figure 3. Northern stain analysis Score 6.5 of expression- And cyclin 1.1 kbses report card D2. RNA of ten microgramses excerpt to respond the MuLV from the different Graffi Tumor( a) or from according to the designation's normal gauze 织品 ( B) is in the top that The path were transfered the nylon thin film and make the 杂 to hand over because of a Cyclin D2 the cDNA fish for to show the report card of 6.5 kbs with a Genomic fragment origins 於 cyclin D2 intron 2 show 1.1 Report card for kb.Notice the cyclin D2 the cDNA to fish for too Show the report card of 1.1 kbs. The size makes the mark's person to be instructions toed theres The patch leaves. Same alike of the stain also were made the 杂 to hand over because of aß- the muscle moves the egg white to fish for. ( a) tumor 7,10, with 11 were acquired from BALB/ c rat, and tumor 4,6,8, were acquired with 12 from the NFS/ N Rat. Tumor 7,8 with 12 were produced by parental Graffi Virus, and tumor 4,6,10, with 11 were produced by GV-1.2 Change. At 3 with 4 it different and latent talent month.
1). 1Were analyzed by northern stain to discover in the tumor to is adjusted atGris1 Were arrange agained and 1 ～相 than tumorGris1Have no quilt to arrange again.Interesting of, emerged the preface row the homology this ET because of 3' endCcnd2 ( the gene's exon 2 with the intron 2). a the specific medicine fish for the origins 於 the quilt report card that show that intron 2 district a 1.1 kbs are strongly to arrange( tumor 6,8, with 12) again in one of overexpressedGris1 quilt three tumorses( diagram3A) compare arrive the tumorGris1 Have no quilt to arrange again, not expresses the information of 1.1 kbs.The expression of the low level discovers in the tumor 4 is this tumor from the 3A 於 fact emergings a nonequimolar DNA to arrange the language again forGris1 ( diagram1A). Tumor atGris1 Were arranges( divided by the tumor 4) agained to also perform the high level the cyclin D2 of normal 6.5 kbses the report card.The report card that AW060277 that that 1.1 kbses match is too disheveled hair until ET is a lot of now than lower level is in the normal gauze 1A 织品 inside( diagram3B). Tallest expression is from the brain, ovary in the thin 3B 纱织 the 品 inside see, long 15 days's embryo brain, with 15 days- Old and whole embryo( diagram3B). 3B
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ET AW060277 that 1 kb that report card change to secret code were sliced the head the cyclin D2 that carry. 4).The game that the preface row that product analyze cDNA show 1.1 kbps in fact is an alternative plan report card cyclin D2 gene. It include the preface row cyclin D2 the exon 1 longer exon of contrast 2( exon 2+) produce the coalescence from the exploitation a the coalescence of possibility other donate the find outing of person's position in the intron district gene( diagram4A).The report card includes an alternative plan 3' shelter the exon( a) signal( AATAAA) of new consistent poly( diagram4B). The openning of cDNA reads what physical stature predict 156 contain the ammonia 基 - The sour protein uses the ～ performance the cyclin D2 to be sliced the head the cyclin that carry into box.Other possibility of coalescence position that usage that sour additional inside is because of 2 results of exons at 20 cloak-and-dagger contain the ammonia 基 first 136 sour cyclin D2 for containing the ammonia 基ss that arrives and precocious terminate that to proteinly be stopped by 於 that TAA of emergence the codon to is morally upright in the remnant 156 its 後 ( diagram4B). 78 And that highly kept the cyclin the box that lack thated most the 23 kinds 4B 78 後 contain the ammonia 基 sour( diagram5). Interesting of, preface row comparison in that its C for preface row for full-lengthly with that slicing the head carrying the cyclin D2 the protein emerging the 5 残 staying the thing protecting the L143, L146, P155, with in the H156 in the district where second protein disagreeing in that cyclin box- The computer terminal ends( diagram5). 5
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Figure 4. The essentials's performance Normal and two 择 a the ground join togethered the cyclin D2 the cDNAs. ( a) thatThe solid box expresses the normal of five exons( Ex 1 arrive-5) Cyclin D2 gene.At two 择 a the report card inside of 1.1 kbs of the ground coalescences, Exons 1 that normal cyclin D2 of similar to 2 is 於 of those, withWere expressed the readtrough district by box hatching Constitute the exon 2+.Were ordered the box that engrave to express the alternative plan exon 3'. ( B) the cDNA 核甘 preface of 1.1 kbs row with that Inferenced to contain the sour preface row of the ammonia 基 . Three exonses beginnings order Quilt arrows designation. In the preface pike on the row head Instruction the report card exon of 6.5 kbs 2 of be over. That Consistent signal of the polyadenylation is a the box pack of.-|, 百拇医药
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Figure 5. Contain the sour comparison of the ammonia 基 Preface row that slice the head to carry( Trc) with the full-length( D2) cyclin D2. First 136 残ss stay what thing is same similar to change to secret code quilt Preface row. An arrows designation of cyclin box at an end quilt.
Gris1 The quilt is to arrange again( diagram6). 3B), also were analyzeds and also showed to be sliced the head the protein's emergence that carried.Control, come from protein that NIH 3 T3 fibroblasts the transfected squeeze out the thing because of the pCMV- D2trc, and expresses the 6 3B 矢 measured the containment the cDNA encoding that to slice the head to carry the protein, and also include the detectable amount to is sliced the head the protein that carry( diagram6).Alike of the result also were acquiredses because of polyclonal 6 浆 the liquid oppose complete rise to slice the head to carry the cyclin D2 the protein.( the data does not show) this 浆 the liquid also cyclin D2 is discoveredses then 34 kDas protein.( the data does not show)4;t$%#(, http://www.100md.com
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Figure 6. That expression is discovered The rat's product is in the NIH 3 T3 fibroblastses inside slice the head to carry the cyclin D2 And the endogenous sliced the head to carry the cyclin D2 the protein. Western stainNormal gauze 织 tumor that the 品 's analysis respond the MuLV with Graffi is Show. Protein is from the adult rat the brain( path 1) quilt excerpt; Ovary( path 2); tumor F6,Gris1 The quilt is to arrange again ( path 3); with there is pCMV- D2trc NIH 3 T3 fibroblasts transfected plasmid.( path 4) protein(30 to 80µg) Were loaded at on SDS-12% polyacrylamide gel electricity the 泳 move the gum 化 with The electrophoretically turns to move to the polyvinylidene difluoride Thin film, and the immunodetection use the antibody to be circulated The cyclin the last 20 kind objection were sliced the head to carry to contain the ammonia 基 sour to rise D2 protein.
7). XenopuslaevisThe cyclin quilt predicts to change to the secret code's D2 cDNA on resembling of sliced the head to carry the cyclin D2(38)( 38 Diagram7).However, these writers do not the 38 7 阐 is clear such of what research imagines that the possible role of a report card, with not further slice the head to carry the cyclin D2 to report. Take together, and these results emerge the alternative plan report card encoding cyclin the D2 to keep because of the quilt of a cyclin box for drive slicing the head carrying at mankind of in, rat, withXenopus2hgz, 百拇医药
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Figure 7.Than that predict Come from the containing of rat the ammonia 基 sour to is sliced the head the cyclin D2 preface row that carry,Xenopus 40).2hgz, 百拇医药
Gris1 There is ability decontroling to express that cyclinD2 gene downstream the ground find outed 85 kbp. Really, the retroviral integrate atGris1Lead to increase the expression cyclin D2 of two report cards the gene: That is already- mean special features encoding that 6.5 kbs that report card have already known 289 contain what ammonia 基 - It is sour and all to grow the protein(19)19And a the report card origins of 1.1 kbs 19 於 coalescence of possibility other is into the code a 156- cyclin D2 containing of protein ammonia 基 - Sour isoform.Decontrol the expression cyclin D2 the gene to also have in the T- cell to is reported the leukemia by the BL/ VL3 to project the line leukemia the virus to respond.(11,41) 114141 We analyze this in the BL/ respond the VL3 tumor 133-1 inside of report card gift with discovers that it in the tumor having as if general mobility of report cardGris12942 Increasing the expression retroviral rule that insert that gene also seem a kind of mechanism of its coalescence of changesed to not yet investigate. Short report card(2.9 with 1.3 kb) the cyclin D2 gene too contain erythroleukemia cell of quilt report at rat section (19)19 And in the cell row of various different person tumors a line(15,24), 1524The possible right and wrong of the gene's rule of that that coalescence of designation is often complicated of. It is knowing that cyclin the D2 regard as the 112942 19 1524 ～ to use the emollient proto- oncogene, and be it to can convert to nominate the primary election of the candidate meeting rat embryo fibroblasts when overexpressed is at the conjunction phrase because of the incitement Hey- Ras(17). 17Often were seen the malignancies in the various different people in the overexpressionD2 affixture, cyclin, for example T- cell lymphoma, testicular cancer, with the ovary's tumor.(2,33,39)23339The report card overexpressed that data present to perform that 1.1 kbs here changes to secret code a the C in the Graffi tumors- sliced the head to carry the cyclinD2 protein in the bitter end. The report card may not too be the information of the aberration, nontranslated, from the protein hereafter that were sliced the head to carry in the anticipant piece 17 kDas clearly discover in the tumorGris1 Were arranges agained and expresses in the normals the report card's gauze 1723339 织 of 1.1 kbs of 品 .Therefore, it is the conceivability's new isoform and also the possible in the light of ～ use the proto- oncogene standing alone of or use the full-length isoform in the cooperation aspect or because of others oncogenes.
D- The type cyclin expresses and during the period of change by the mitogens to respond G1- Cell period of to- The S is period(24,25,32). These respond D-The type cyclins becomes the synthesis because of the cyclin- Dependent kinases ( Cdk-4 is with the Cdk-6) through that highly kept the cyclin boxed(3,23, 27). Is°which arrive proof the phosphorylate pRb, and suggest a role recently For the rule of that synthesis at G all0- arrive- G1 Period The change is with rule that grow in the cell in the cell Circulation go forward(5,9,26). The cyclin D2 new isoform identifies Is a C here- In the protein that the bitter end sliced the head to carry to lack 133 contain what ammonia 基 Long isoform of sour, include the 23 kinds the last to contain the ammonia 基 sour From the cyclin box, but take 20 kinds contain the ammonia 基 sour and new to stretch. Whether cyclin D2 that we not and still know is sliced the head to carry or not is still retention Its bind the Cdks ability.However, the short isoform present Preface row is in this district of have to grow the aegis of the isoform 20 The 残 stays the thing in the 残 to stay the thing the L143, L146, P155 with the H156, only at Cyclin at an end box, suggest these 残s to stay the thing can There is the importance's function role, perhaps in the formation aspect There is Cdks a synthesis or because of other hand over with each other function partnership. Recent of proof to Cdk-The neutral sends to point out the making of D- type the activity Cyclins. Really, generally show the mutual influence of cyclin D2 because of DMP1, have to resemble the similar repetition protein of Myb, through its carboxyl- computer terminal Three of half( the 残 stay the thing 142 to 253) and that is not cyclin of D/ Cdk/ DMP1 The synthesis can be discovered(13). Because the DMP1 can't affect enough and each other, later on does not shelter the district from it Importance of for bind.
Show such as the analysis of our northern stain, the report card of 6.5 kbs Cyclin D2 the gene expresses in the high level quilts at respond the virus Tumor, not have at thoseGris1 The quilt is to arrange again, with At a thin 纱织 of nontransformed the 品 's variety. More and more limit Style expression that 1.1 kbs that report card instructions that Wering sliced to go to the head of the cyclin to carry can play a role in the development Thin 纱织 of specific medicine the brain that 品 resemble is a big part with the ovary, it Often expresses. D2 gene( because of both all that 6.5- And the report card of 1.1 kbs disrupt) Propagating of granulosa for emerging the weakness in that ovary Cell is with the granule with the stellate cerebellum interneurons (33,14). Report card, it think deeply to be sliced the head the cyclin that carry in the come-on The D2 can also have a role in these cells to type.u!:|s*d, http://www.100md.com
The cyclin family other member has the quilt to show two 择 a the ground have The coalescence report card predicts to slice the head to carry the protein. Such report card Have in the tumor cell the capable cyclin E is reported(18,28) And because of in the cyclin in various normals the D1 line up with tumor cell a line (4). however, two report card that 择 a the ground join together to describe Because the cyclinD1 and ～ is different from is discovers for cyclin D2 of those, When they change to secret code a the protein slice the head to carry in the little tiny part C-Computer terminal, be over.In the affixture,1 2 择 a ground coalescence The report card cheats to a to compare the isoform of the shorter cyclin C because everything go to the head to carry The cyclin box also is reported(22). Compare the shorter cyclin isoforms the energy to play the important role of the cell rule, At least in some specific medicines thin 纱织品 . Function of to the point Were sliced to go to the head of the cyclin D2 to carry to support to were appeared by that two 择 a ground Join togethered the information to cheat to for slicing go to the head the database of cyclin D2 at ET that carry the inside Because person is with the representative special features quilt Taieb with Jessus(38) atX. laevis.
We do not know to be sliced the head the isoform function role that carry In the normal cyclin in the cell D2, but we are in the inquisition its expression Pass by the cell period with particular hand over with each other function partnership Cloak-and-dagger protein.8^3%7s[, 百拇医药
This work drive the bestowal of Cancer from nation 007072 support Academic association Canada with award by the academic association from Canada 37994 Health research. The C.D. is what Cancer study to accept Social company studentship is with the FCAR Ph.D. Scholarship.8^3%7s[, 百拇医药
Adamson, M. C., J. 镀 is with the silver, with the C. A. Kozak. 1991. ThatCatch the rat gibbon ape leukemia the virus to suffer the container equal or the homology's thing: The 映 that inherit the gene shoots to act with container of suffering of a possibility in the rat. 滤 is over the sex microbiology183:778-781.8^3%7s[, 百拇医药
Bartkova,J.,E. Rajpert- De Meyts, N. E. Skakkebaek is with the J. 1999. At the adult mankind testis with the D in the testicular- Type cyclins Cancer: Relate to the cell type, and propagate, distinct, with Malignancy. J. Pathol. 187:573-581.
Die down, S., L. Bonetta, D. MacAllan, D. Fend off, A. Holder, C. 1994. The CDK6( PLSTIRE) is on using the cyclin consociation cyclin with the CDK4( PSK- J3)- Counting on that kinases that clear son gather D1. Oncogene9:71-79.'!2*{, http://www.100md.com
Betticher, D. C., N. 茸 , H. J. Altermatt, P. Hoban, W. 1995. The agent coalescence livings the product a novel Cyclin D1 report card. Oncogene11:1005-1011.'!2*{, http://www.100md.com
Datar, S. A., H. W. man, A. F. de la Cruz, C. F. Lehner, And B. A. Edgar. 2000. That果蝇 cyclin D- Cdk4 synthesis Promote the cell's growth. EMBO J. 19:4543-4554.'!2*{, http://www.100md.com
Denicourt, C., E. Edouard, with the E. Rassart. 1999. Oncogene In the Graffi rat the section's leukemia virus proviral myeloid leukemia making of inside activity Integrate. J. Virol. 73:4439-4442.'!2*{, http://www.100md.com
Deer of idle woman, S. F., P. W. 雌 , K. Louie, S. I. 芦苇 , A. 阿诺 , And R. A. Weinberg. 1993. Retinoblastoma of physically hand over with each other function Someone or other D cyclins protein. Cell73:499-511.'!2*{, http://www.100md.com
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Huard, J. M., C. C. Forster, M. L. Carter, P. Sicinski, with M. E. Ross. 1999. The Cerebellar histogenesis were lacked by disturbance in the rat of Cyclin D2. Development126:1927-1935.
Inaba, T., H. Matsushime, M. Valentine's Day, M. F. Roussel, C. J. Sherr, with the A. T. facial expression. 1992.Genomic buildup, chromosome of Confine, with the gene's independent expression of cyclin D that mankind. Genomics13:565-574.@v, http://www.100md.com
Jonkers, J., with the A. 伯恩 . 1996. The Retroviral insertionalmutagenesis is as if to identify a strategy of the cancer gene. Biochim. Biophys. Acta1287:29-57.@v, http://www.100md.com
Kerkhoff, E., with the E. B. Ziff. 1995. Cyclin D2 is with the Hey- Ras Converted the rat embryo the fibroblasts to exhibit the 品 cell unusual to decontrol the size The S period control arrest with early in the low 浆 liquid. EMBO J. 14:1892-1903.@v, http://www.100md.com
Keyomarsi, K., D. Conte, two a life times, W. Toyofuku are with the M. P. 福 gram 斯 . 11:941-950.@v, http://www.100md.com
Kiyokawa, H., X. Busquets, C. T. 鲍威尔 , L.Not government buildup, R. A. Rifkind, with the P. A. mark. 1992. come from the rat section the animal's a D- The replication of the type cyclin Erythroleukemia cell. Proc. Natl. Acad. Sci. The United States89:2444-2447.
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Lammie, G. A., R. Smith, J. Silver, S. Brookes, C. Dickson, And G. Peter. 1992. In the rat of the Proviral near the cyclin D1 insert lymphomas: For the B of BCL1 translocations at mankind- Cell parallelism in the neoplasms. 7:2381-2387.;foc|&, 百拇医药
Lee, H., J. M. Lahti, with the V. J. Kidd. 1996. Two 择 a groundJoin together the cyclin C the mRNA to broadly is expressed, and the cell period manage, and change to secret code to is sliced the head the cyclin that carry into box. Oncogene13:705-712.;foc|&, 百拇医药
Matsushime, H., M. E. Ewen, D. K. Strom, J. Y. Kato, S. K. 汉 gram, M. F. Roussel is with the C. J. Sherr. 1992. Confirm with Be unsuited to the property that catalytic subunit( p34PSK- J3/ cdk4) of type is the mandible's D Key-in the G1 cyclins. Cell71:323-334.;foc|&, 百拇医药
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Received the 2002 July 3/ the 2002 of the general acknowledgement September 16'k, http://www.100md.com
68:540-558,1957). Gris1 ( integrate the position because of the Graffi 1). 滤 is over the sex poison the 菌 's integration atGris1 Disheveled hair now 13% of tumor analysis. ThatGris1 The place was shot there end the department the district for of rat chromosome 6 by 映 ,85 kbs heading up to swim that cyclin D2 the gene. Gris1 Major cyclin that cause 6.5 kbs of that normals of overexpressions report card that report card that D2 is besides too outside lure that new expression, two 择 a the ground join together 1.1 kbs the cyclin D2 gene that change from that to the secret code everything the head to carry 17 kDa of cyclin D2. The report card's expression of that this 1.1 kbs is a particular tumor to ～Gris1'k, http://www.100md.com
16 44). 44
Graffi complicated 滤 is over the sex poison the 菌 is the first from have no the cell insulation 滤 liquid Ehrlich tumor with almost exclusive respond the myeloid Leukemia when in the infant that rat that some strains inoculate (10). Is from that 滤 over the special features that sex poison the 菌 's synthesis insulation and(30). The replication right and wrong of these two numerators often resembleses in the construction, and apart from Make duplicate the GV-1.2 to lure a short delitescence of disease with ～ Period with in the U3 performance in the enhancer the duplicate of 60 bps of a perfections District that hope earnestlies computer terminal repetition(( LTR)30). New-born BALB/ c And the NFS rat inoculate with one of the replication of two numeratorses Or because that parental admixture develop, to different degree, The hepatosplenomegaly, thymic extended the language, generalization the lymphadenopathy, And present to public in the inside's anemic leukemic.So that, Graffi rat section animal The virus( MuLV) of right research leukemia were considered a good model Myeloid mechanism leukemia inductive method with go forward And arrive to recognize the clear and cloak-and-dagger proto- oncogenes.
The Graffi virus lures the leukemia's accurate mechanism Is not understanding that like. However, making of the cell's proto- oncogenes activity Quilt insertional the mutagenesis is a main step in the leukemic procedure. We in times before have to report the making of c the activity-myc,Fli1,Pim1, And Spi1/PU.1Integration of Graffi virus gene at 20,10,3.3, And 3.3%, respectively, tumor test(6). The in order to identifies the new cell to proto- oncogenes, we contain replication with Meant an unusual 滤 that come from the Graffi over the integration position that sex poison 菌 that the special features respond the MuLV Tumor.6w-3&, http://www.100md.com
In this research, we report confirm with describe Gris1( because the Graffi integration make 1 locate), a new usually position Respond the MuLV integration in the tumor in the Graffi. 滤 is over the sex poison the 菌 's integration AtGris1 Gris1 The place was shot the rat chromosome by 映 6 of the district of the end department And were located to face the upper stream cyclin D2 the gene of 85 kbp. That 滤 is over the sex poison 菌 of Integrate atGris1 Cyclin main report card D2 gene and incitement expression New, still- Uncharacterized alternative plan the report card of 1.1 kbs From the homology's gene.
Main of tumor. NFS/ N is with the BALB/ c the rat were purchased from Frederick Cancer to study the equipments, Frederick, Md., with from check the 尔斯 river the company, St. Constant, Quebec, Canada, respectively. The freshman's rat(<48H past) about use what～ inoculate the intraperitoneally105 PFU at 0.1 to 0.2 毫s rose the percolation 滤 the sex poison the 菌 's 浮 at superficial.k63{ie, 百拇医药
Genomic- DNA with- RNA analysis. High mark son- The weight genomicDNA were excerpted from the congelation Normal is with the thin 纱织 of leukemic 品 quilt according to the standard procedure that describe In times before(31). µGenomic g Come from what tumor of the DNA or control organ is digested because of an adequacy Limit the endonuclease, and separate on first 0.7% the agarose gel, with Transfer a nylon thin film.( Osmonics)k63{ie, 百拇医药
Total amount RNA is from the frozen normal with the thin 纱织 of leukemic the 品 were excerpted Because of the TRIzol reagent( life technique) according to that The instruction makes the operator. Analyze to 於 north,20 µThe g of the total RNA at 1.2% A 荃 agarose on were separated According to the gel that describe in times before(34) and transfer the nylon Thin film.( Osmonics)
All stain at 50% formamide-10% the 士 get the 郎 the sulfate-0.5% the inside is made the 杂 to hand over 钠 dodecyl sulfate( SDS)-1 the M NaCl is then 42 ° C because that is appropriate Fish for. By arbitrarily of the handbook extension perform the lifelike usage oligohexamers( Pharmacia P- L biochemistry of, Montreal, Canada). The thin film were washed At 2x x SSC is 0.15 sour salt of 檬 of M NaCl-0.015 M 钠柠 ) for 30 The least is then 42 ° C with at 0.2x^!\?, http://www.100md.com
滤 is over the sex poison the inserting of 菌 make enlarge to locate. Enlarge that provirus- The cell's DNA 联 connects to be circulated By the PCR(37). Usage of extending specific medicine of a kind of biotinylated handbook is that Graffi U3 district that LTR(5' AGGCGCAAGGTCGTTTCAGGTCTTTGGG 3') with 1 5 partially deteriorated drive handbook that describe( FP) In times before(36). In the streptavidin glazed on magnetism grain son.(Boehringer Mannheim) The second PCR is then in the recovered biotinylated on circulate Amplicon usage a 筑 nest Graffi virus- Specific medicine handbook(5' ATGGGTCTCTTGAAACTGCTGAGGG 3') with on matching the nondegenerated handbook allotment FP handbook is in the first PCR convenient(5' CAGTTCAAGCTTGTCCAGGAATTC 3'). Because of the tumor 6 with 8, at the 滤 in the district over the sex poison the 菌 's integration at Gris1 AndCcnd2 The specific medicine is that genomic district atGris1 AndCcnd2 ( a The handbook is each 10 kbp) in the combination one of convenient two handbookses The specific medicine is a Graffi LTR. The handbook specific medicine is a Graffi The LTR is a Graffi 254-229(5' GGGGCAACCTGGAAACATCTGATGGG 3') with Graffi 10-34.(5'CCCCACCATAAGGCTTAGCAAGCTAG 3') genomic The specific medicine handbook designs the winnings according to the preface row In the Ensembl rat the genome wait upon the 器 To 於 chromosome 6 district 128.05 to 128.15 Mb. Handbook Is according to following each item: GRIS1F,5'GTCTCTTTTGTTGCTTTGAGGGGC 3';GRIS2F, 5'GAGAAGGTGAATGGTTTTTATGCCC 3';GRIS3F,5'CAGAGTTAGAACCTGGGTGTGTGC 3'; GRIS4F,5' CTGACAAGCCGCCCTAGGTG 3'; GRIS5F,5' GTCCATTTTCCAGGCAGGACTGAG 3'; GRIS6F,5' CTGAGCCCTCCATATCAGTCATCA 3'; GRIS7F,5' TTGATTTTTGCCTGCTTGTCCTCAC 3'; GRIS8F,5' TCTTCCACTTCCTGGTTTGGTATC 3'; GRIS9F,5'CAGGAGTAACGGGAGAGAGCTA 3'; with the GRIS10F,5' TGCGTTAAGAGGACTGAGACCC 3'.
The 映 that inherit the gene shoots. Gris1The southern stain 映 of quilt shoots two combinations's analysises inherit the gene of The cross: ( NFS/ N or C58/ Jx The 12 musculus of the 12 Greek alphabet of Greek alphabet)F1 x M. (20) and( NFS/ Nx The 12 spretus of Greek alphabet)x M. spretus Or C58/ J(1). Descendant These the crosses have to be typed because exceed 1,200 person's allotments that make the marks Exceed to have of 19 autosomes with X chromosome. Combine the distance Decide the recombinants from the arithmetic figure quilt, and loci is By reduce the minimal order the arithmetic figure to reunite.|]?r), http://www.100md.com
Genomic- The replication isolates.The BAC genomic make duplicates to include thatGris1 By the reviewing RPCI-129 S6 part 2 rat BAC library( BACPAC Resources; CHORI, California Auckland.) Because ofGris1|]?r), http://www.100md.com
Case shot hunting rifle preface row.Case shot hunting rifle the library quilt construct to consider as to in times before describe (43). Brief, the BAC DNA is a large-scale alkaline preparation of quilt The symptoms that there is the big reduces the 退 structure the material. (Qiagen) µg) Does the sonicated is a nuclease to fix the buffer inside in the mung bean son(30 毫莫 ear 钠 Acetate,50 毫莫 ear NaCl,1 毫莫 ear ZnCl2,5%glycerol). Fragment be over Then 30 ° Cs are to have 40 U of 30 leasts is fixed the mung bean the son nuclease.(Amersham- The Pharmacia livings the 化 technology) the sample is an electrophoresed At 1% agarose on gum 化 , with 800- Fragment for 1,200 bps, is a gum 化 The 洁 is clean to enter that with ligated insideEcoThe recreational vehicle make duplicates the position pBlueScript 2 KS(+).( Stratagene) the tubal ligation product is a conversion Enter to use the Dh5 of the chemistry ability inside{alpha} The alkali symptoms that DNA replication were prepared was reduced the 退 by small scale because of The Miniprep equips from the Qiagens. That preface the row respond is Use big- 颜料 terminator the period circulate the preface row the material the edition 2.0( applied Biosystems, California city that keep and raise.). Respond to be involved a model number 3700 DNA analysis.( applied Biosystems)
Game. The cDNA ends( game) to extend quick 5' with 3' experiment Were circulated to use the GeneRacer material. ( Invitrogen) that game- prepare the good appearance The cDNA pond were acquired the usage the GeneRacer 寡 the 醣 ( dT) handbook On the tumor, total RNA of F6. 5 Of enlarge' end to express Preface row the label( ET) AW060277 is a finished usage that GeneRacer 5' handbook with ET- Specific medicine handbook 5' GTCTTGGTTAGTGTGGCGGCCTTA 3'. GeneRacer 3' handbook with that ET- Specific medicine handbook 5' TCACACTAAGGCCGCCACACACTAA 3' the habit 於 acquire 3' end.-yallkp, http://www.100md.com
The cell cultivates the circumstance, plasmid building with the transfections.NIH 3 Dulbecco's of T3 cells at correction eagles were supported The medium( life technique) uses 10% calf 浆 liquid complement ( life technique). pCMV-The D2trc constructs the quilt the replications to do The beguilement quilt that that is complete acquires of is sliced the head the cyclin D2 preface row that carry By the PCR because of following and various handbooks: 5'TTTCACCCTCTAGAATTTCCATG 3' with 5' AGGAGGTAAGGGAAGCTTTCC 3'. The fragment is a replication At thatHindIII-XbaThe position wireless of my pRcCMV inducement. (Invitrogen) The Transfections uses to be done the Polyfect( Qiagen) according to The instruction of the manufacturing operator.
The antibody preparation, protein draw outs with the western person's stain. The last 20 kind that come from the C- computer terminal of protein and objection contain the ammonia 基 sour The be over produces. Brief, right should of cDNA preface row Is a PCR to use the glutathione in the physical stature to extend and make duplicate S- The tranferase enters pGEX-4 Ts-1ses the protein in the vector.(Amersham Biosciences) After inductive method, bacterially squeeze out the thing to is delivered the glutathione- Sepharose 4 protein 洁 that B column( Amersham Biosciences), with were made clean Is a skin bottom of into New Zealand white rabbit inside inject. The 浆 liquid collected immediately after of 凝 piece the 缩 enter and store at -70 ° C. NIH 3 T3s contain pCMV- D2trc fibroblasts transfected The plasmid were washed to twice there is ice in the RIPA- A cold PBS is with the lysed Buffer(0.15 M NaCl,1% NP-40,0.5% 钠 deoxycholate,0.1% SDS,0.05 M Tris- HCl[ pH 8]) with complete the protease depressant Chicken tail wine( the Roche applied science) for 30 then 4 ° C. The protein is minimum Come from the squeezing of tumor out the thing with the thin 纱织 the 品 quilt prepare in the SDS-The urea buffer is inside (0.5% SDS,8 M urea). The protein's concentration at that is different Squeeze outing thing to is measured to use to living the 化 Rad the protein to analyze. Opponent Lysate protein quantity(30 to 80µg) Dilute At 2x The 抽 takes the sample buffer(30% glycerol,4% SDS,160 毫莫 ear Tris- HCl[pH 6.8],10%ß- mercaptoethanol, with 0.02% bromophenol 蓝色 of) with boil the 沸 to 3 the 最 ? the The protein is separately at 12% on SDS- Polyacrylamide gum 化 electricity the 泳 move with the electrophoretically Turn to move to the Immobilon- P polyvinylidenedifluoride thin film (Millipore). The thin film were jamed to include in the PBS 0.2% Tween 20 with 4% 浮渣 milk powder( PBS- Tween- milk) for 1 h at Room 温 . Incubation because that make the versa 洁 clean slice the head to carry Sour antibody of ammonia of cyclin D2 缩 (1:100 in the dilution in the PBS- Tween- milk) Or the anti slice the head to carry the cyclin D2 浆 the liquid( in the PBS- Tween- milk of 1:5,000 dilution) Is for 1 h is in the room 温 movement. In the incubation's 後 , The stain includes in the PBSs 0.2% Tween 20 insides is washed( PBS- Tween) And embrace for have the versa rabbit immunoglobulin G the peroxidase of 1 h Combination.(1:5,000 in the PBS- Tween- milk)( BD Pharmingen) that is immune Of the synthesis were shown the usages the ECLs plus chemiluminescence Reagent.( AmershamBiosciences)
ConfirmGris1, in the position that the integration in the Graffi is a to usually respond the MuLV tumor.Identify the new proto that stimulate of quilt- The oncogenes responds the MuLV tumor in the Graffis, drive Sorensen PCR method that describe is with the colleague(37)37Were applied the 37 於 the winning the 格状 to automate the DNA to attackstone the on the side 5' come from the tumor 12 of Graffi proviruses be over. A the special genomicPCR fish for 400 bps is produced and used to investigate from that tumor 30 of other tumor DNA of patch. On the southern stain, this fish for( fish for a[ diagram1B]) can quilt is discovered then the alignment's allele is in the tumor 12 DNA inside and at an other tumor( tumor 4) of inside( diagram1A).More the low quilt is again normal allele of the alignment's allele mightiness and ～相 is in the tumor 4 suggest that 滤ed the sex poison the 菌 's integration at this position for that tumor partially is a clonal.Two proviruseses both are all at the homology in that place of settle the directions aspect and is located besides each other outside of 1 kbp. Gris1.kn, http://www.100md.com
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Figure 1. Southern stain analysis The Graffi responds the MuLV 的白血 ? the ( a) normal spleen is with the tumor DNA Digest because ofCheat嗨 with were fish fored on make the 杂 hand overing. (B) Limit the map thatGris1 Place. Position with Settling of proviruses directions quilt arrows designation.映 that that fish for and used to discover then arranging the language and because the heredity gene shoot isManifestation is such as the solid box. ( a with the B, respectively) fish for an is The quilt PCR acquires, and fish for the B origins 於 genomic DNALambda the replication. The abbreviation is to limit the endonucleases: E,EcoRI; B,Cheat嗨 ; R,EcoRecreational vehicle ; S,SacI; P,PstI; X,XbaI. ThatXbaMy fragment is a length 13 kbp.
Gris1 Fish for on were acquired by PCRing to use to investigate the genomicLambda to come from the rat the phage library of the 129/ Sv the winning the replication to express that germs the lineGris1 Place. The replication of a 8 kbses is a recovereds with use to construct a restrict map of amenity with get from a long afterwards is used special to fish for( fish for the B) to shoot for 映 of inheritting the gene( diagram1B). 1B:xcf#ch, 百拇医药
The 映 that inherit the gene shootsGris1.Decide that the heredity the gene- Map positionGris1, we investigate Inherit the cross in heredity gene of two combinations is inside of this make the mark's person. Combine the inside the cross, southern stain the analysis to identify in the first 6.8 Kb ofEcoRI fragment atM. M. musculus And the fragment of 5.2 kbs In the NFS/ N rat.Combine the inside in the cross's the second,Sac2 Digest Produce the fragment of 3.0 kbs atM. spretus And the fragment of 1.2 kbs In the NFS/ N rat. The style of the different fragment heredity At those two the crosses's combination quilt and ～相 than 1,200 of The person who make the mark in times before typeses and the 映 shoot. The linkage is observed because of Make the mark's chromosome of person at on end department 6. Gene order of sequence is with the recombinational The distance is according to following each item: Raf1 (3.0 ± 1.1)-Tnfr1 Ltbr (0.4 ± 0.4)-Scnn1a Gris1 Ccnd2 (5.2 ± 1.5)-Kras2.
Quilt 滤 is over the sex poison the gene that the 菌 's integration stimulate confirm atGris1. Or by the 滤 over the sex poison the 菌 's integration bluntness 化 atGris1 That 8 kbs ofGris1 The genomic Lambda is the expression's preface row replication. However, have no a transcriptional unit can quilt Northerner detection Of stain analysis.( the data does not show) generally and widely behaves to know the gene to make the activity Integration that quilt 滤 is over the sex poison the 菌 exceed the big distance can take place(12,21, 35).The so that, we searches the candidate gene at a district About and about 200 kbp environments thatGris1Insert to locate. Contig thatGris1 The district were acquired a rat by the reviewing 129/ Sv genomic BAC library. Two hand over the 叠 to handle the BAC replication is Recovered and used to produce the district's a restrict map ( diagram2). The most long replication recovered is in the sample case shot the hunting rifle inside were used The preface row designs to acquire the preface row of a 2.5 fold layerses the surplus 200 District for kbpGris1. The each preface row then were analyzed Personally oppose the NR, decibel with the HTGS 核甘 database Use the fresh breeze 运 the calculate way then. We preface row is discoversed same alike at that Database because of two already gene that know and because some ETs are irrelevant Arrive already gene that know.Those two already gene that know is for the triose phosphate is those isomerase(Tpi) and cyclin D2(Ccnd2). Position that Comparison of two genes arriveGris1 ( diagram2). TpiThe gene heading ups to swim to about is placed 30 kb Gris1, however thatCcnd2The gene about were placed 85 kbp Downstream ground( diagram2).Decide whether to express that or notTpi And Ccnd2 Gene quilt 滤 is over the sex poison the 菌 's integration changes atGris1, Similar to anyly present of genomic fragmentTpi OrCcnd2 Were used in the northern stain analysis. Even we Place thatGris1 The place has 30 kbp fromTpi, did not change In the report card's expression description of the gene's 1.5 kbses Can be a convenient 滤 of tumor in integration hair over the sex poison 菌 nowGris1 Comparison because of the other Graffi tumor with control the organ( the data is not Show). Two tumorGris1 Were arranges agained( tumor 4 with 12) Emerge in the anticipant size normal cyclin D2 information 6.5 Kb, but amazingly of, two tumors present an affixture of The band of the mightiness's 1.1 kbses( diagram3A). This new cyclin D2- Specific medicine band Is in the tumor absentGris1 Have no quilt to arrange again( tumor 7,10, with 11). Tumor 6 with 8 also present a mightiness of 1.1 kb of Band, even our must not detection the DNA arrange the language again at ThatGris1There is place that fish for an and B. Later on locate from the integration Were located about 85 kbps keep off the cyclin D2 the gene, we the deep thought That tumor 6 with 8 most and probably sheltered a 滤 the sex poison the 菌 's integration It is some to be placed inGris1 Fish for a with the boundary that the B discovers. Have already take placed at thatGris1 Extended the 6 different from 8 because of the handbook of doubles tumor, a Specific medicine is because of the Graffi MuLV U3 LTR and another origins 於 Each 10 of kbps at cyclin D2 gene with thatGris1 Place. This procedure also is circulated because of the U3 and genomic handbook right Match the another retroviral to settle the directions. PCR product Use a kind of U3 LTR- The specific medicine were made the 杂 to hand over to fish for. According to expect, we Can a special U3 is discovered- The specific medicine fragment is each tumor because of Combination for there is handbook( the result not show)ing, certain Provirus emergence at thatGris1 Place is in the tumor 6 with 8 Then 4 with 5.5 kbp, respectively, heading up to swim thatXbaMy position 5 Is which' the detection boundary of the first analysis. That Provirus emergence at thatGris1 In the tumor 6 insides's place with 8 is Also analyze the proof the usage by the southern stain three different Get from the genomic to fish for. ( consequently not show) consider as the decision quilt Proviruses PCR, that come from the tumor 6 similar to 8 is atly settle the directions aspect Consider as those in the tumor 4 with 12 inside.So that, outlying 4 tumorses 30(13%) Present a the Graffi MuLV integrate at thatGris1Place is with the expression A the cyclin D2 of new 1.1 kbses- Specific medicine mRNA.
fig.ommitteed/m0l49, 百拇医药
Figure 2. Predicted the actual map ThatGris1 Genomic district. The BAC uses the ～ replication the recovered thatGris1 MluI-,ClaI-, withPvuI- Digest the BAC replication. On the map of the small arrows instructions that 滤 is over the sex poison the 菌 's integration position with settle the directions to tumor 6 With 8 with the tumor 4 with 12. ThatGris1 Use of fish for is Show and have no quilt painting according to the comparison to decide./m0l49, 百拇医药
fig.ommitteed/m0l49, 百拇医药
Figure 3. Northern stain analysis Score 6.5 of expression- And cyclin 1.1 kbses report card D2. RNA of ten microgramses excerpt to respond the MuLV from the different Graffi Tumor( a) or from according to the designation's normal gauze 织品 ( B) is in the top that The path were transfered the nylon thin film and make the 杂 to hand over because of a Cyclin D2 the cDNA fish for to show the report card of 6.5 kbs with a Genomic fragment origins 於 cyclin D2 intron 2 show 1.1 Report card for kb.Notice the cyclin D2 the cDNA to fish for too Show the report card of 1.1 kbs. The size makes the mark's person to be instructions toed theres The patch leaves. Same alike of the stain also were made the 杂 to hand over because of aß- the muscle moves the egg white to fish for. ( a) tumor 7,10, with 11 were acquired from BALB/ c rat, and tumor 4,6,8, were acquired with 12 from the NFS/ N Rat. Tumor 7,8 with 12 were produced by parental Graffi Virus, and tumor 4,6,10, with 11 were produced by GV-1.2 Change. At 3 with 4 it different and latent talent month.
1). 1Were analyzed by northern stain to discover in the tumor to is adjusted atGris1 Were arrange agained and 1 ～相 than tumorGris1Have no quilt to arrange again.Interesting of, emerged the preface row the homology this ET because of 3' endCcnd2 ( the gene's exon 2 with the intron 2). a the specific medicine fish for the origins 於 the quilt report card that show that intron 2 district a 1.1 kbs are strongly to arrange( tumor 6,8, with 12) again in one of overexpressedGris1 quilt three tumorses( diagram3A) compare arrive the tumorGris1 Have no quilt to arrange again, not expresses the information of 1.1 kbs.The expression of the low level discovers in the tumor 4 is this tumor from the 3A 於 fact emergings a nonequimolar DNA to arrange the language again forGris1 ( diagram1A). Tumor atGris1 Were arranges( divided by the tumor 4) agained to also perform the high level the cyclin D2 of normal 6.5 kbses the report card.The report card that AW060277 that that 1.1 kbses match is too disheveled hair until ET is a lot of now than lower level is in the normal gauze 1A 织品 inside( diagram3B). Tallest expression is from the brain, ovary in the thin 3B 纱织 the 品 inside see, long 15 days's embryo brain, with 15 days- Old and whole embryo( diagram3B). 3B
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ET AW060277 that 1 kb that report card change to secret code were sliced the head the cyclin D2 that carry. 4).The game that the preface row that product analyze cDNA show 1.1 kbps in fact is an alternative plan report card cyclin D2 gene. It include the preface row cyclin D2 the exon 1 longer exon of contrast 2( exon 2+) produce the coalescence from the exploitation a the coalescence of possibility other donate the find outing of person's position in the intron district gene( diagram4A).The report card includes an alternative plan 3' shelter the exon( a) signal( AATAAA) of new consistent poly( diagram4B). The openning of cDNA reads what physical stature predict 156 contain the ammonia 基 - The sour protein uses the ～ performance the cyclin D2 to be sliced the head the cyclin that carry into box.Other possibility of coalescence position that usage that sour additional inside is because of 2 results of exons at 20 cloak-and-dagger contain the ammonia 基 first 136 sour cyclin D2 for containing the ammonia 基ss that arrives and precocious terminate that to proteinly be stopped by 於 that TAA of emergence the codon to is morally upright in the remnant 156 its 後 ( diagram4B). 78 And that highly kept the cyclin the box that lack thated most the 23 kinds 4B 78 後 contain the ammonia 基 sour( diagram5). Interesting of, preface row comparison in that its C for preface row for full-lengthly with that slicing the head carrying the cyclin D2 the protein emerging the 5 残 staying the thing protecting the L143, L146, P155, with in the H156 in the district where second protein disagreeing in that cyclin box- The computer terminal ends( diagram5). 5
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Figure 4. The essentials's performance Normal and two 择 a the ground join togethered the cyclin D2 the cDNAs. ( a) thatThe solid box expresses the normal of five exons( Ex 1 arrive-5) Cyclin D2 gene.At two 择 a the report card inside of 1.1 kbs of the ground coalescences, Exons 1 that normal cyclin D2 of similar to 2 is 於 of those, withWere expressed the readtrough district by box hatching Constitute the exon 2+.Were ordered the box that engrave to express the alternative plan exon 3'. ( B) the cDNA 核甘 preface of 1.1 kbs row with that Inferenced to contain the sour preface row of the ammonia 基 . Three exonses beginnings order Quilt arrows designation. In the preface pike on the row head Instruction the report card exon of 6.5 kbs 2 of be over. That Consistent signal of the polyadenylation is a the box pack of.-|, 百拇医药
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Figure 5. Contain the sour comparison of the ammonia 基 Preface row that slice the head to carry( Trc) with the full-length( D2) cyclin D2. First 136 残ss stay what thing is same similar to change to secret code quilt Preface row. An arrows designation of cyclin box at an end quilt.
Gris1 The quilt is to arrange again( diagram6). 3B), also were analyzeds and also showed to be sliced the head the protein's emergence that carried.Control, come from protein that NIH 3 T3 fibroblasts the transfected squeeze out the thing because of the pCMV- D2trc, and expresses the 6 3B 矢 measured the containment the cDNA encoding that to slice the head to carry the protein, and also include the detectable amount to is sliced the head the protein that carry( diagram6).Alike of the result also were acquiredses because of polyclonal 6 浆 the liquid oppose complete rise to slice the head to carry the cyclin D2 the protein.( the data does not show) this 浆 the liquid also cyclin D2 is discoveredses then 34 kDas protein.( the data does not show)4;t$%#(, http://www.100md.com
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Figure 6. That expression is discovered The rat's product is in the NIH 3 T3 fibroblastses inside slice the head to carry the cyclin D2 And the endogenous sliced the head to carry the cyclin D2 the protein. Western stainNormal gauze 织 tumor that the 品 's analysis respond the MuLV with Graffi is Show. Protein is from the adult rat the brain( path 1) quilt excerpt; Ovary( path 2); tumor F6,Gris1 The quilt is to arrange again ( path 3); with there is pCMV- D2trc NIH 3 T3 fibroblasts transfected plasmid.( path 4) protein(30 to 80µg) Were loaded at on SDS-12% polyacrylamide gel electricity the 泳 move the gum 化 with The electrophoretically turns to move to the polyvinylidene difluoride Thin film, and the immunodetection use the antibody to be circulated The cyclin the last 20 kind objection were sliced the head to carry to contain the ammonia 基 sour to rise D2 protein.
7). XenopuslaevisThe cyclin quilt predicts to change to the secret code's D2 cDNA on resembling of sliced the head to carry the cyclin D2(38)( 38 Diagram7).However, these writers do not the 38 7 阐 is clear such of what research imagines that the possible role of a report card, with not further slice the head to carry the cyclin D2 to report. Take together, and these results emerge the alternative plan report card encoding cyclin the D2 to keep because of the quilt of a cyclin box for drive slicing the head carrying at mankind of in, rat, withXenopus2hgz, 百拇医药
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Figure 7.Than that predict Come from the containing of rat the ammonia 基 sour to is sliced the head the cyclin D2 preface row that carry,Xenopus 40).2hgz, 百拇医药
Gris1 There is ability decontroling to express that cyclinD2 gene downstream the ground find outed 85 kbp. Really, the retroviral integrate atGris1Lead to increase the expression cyclin D2 of two report cards the gene: That is already- mean special features encoding that 6.5 kbs that report card have already known 289 contain what ammonia 基 - It is sour and all to grow the protein(19)19And a the report card origins of 1.1 kbs 19 於 coalescence of possibility other is into the code a 156- cyclin D2 containing of protein ammonia 基 - Sour isoform.Decontrol the expression cyclin D2 the gene to also have in the T- cell to is reported the leukemia by the BL/ VL3 to project the line leukemia the virus to respond.(11,41) 114141 We analyze this in the BL/ respond the VL3 tumor 133-1 inside of report card gift with discovers that it in the tumor having as if general mobility of report cardGris12942 Increasing the expression retroviral rule that insert that gene also seem a kind of mechanism of its coalescence of changesed to not yet investigate. Short report card(2.9 with 1.3 kb) the cyclin D2 gene too contain erythroleukemia cell of quilt report at rat section (19)19 And in the cell row of various different person tumors a line(15,24), 1524The possible right and wrong of the gene's rule of that that coalescence of designation is often complicated of. It is knowing that cyclin the D2 regard as the 112942 19 1524 ～ to use the emollient proto- oncogene, and be it to can convert to nominate the primary election of the candidate meeting rat embryo fibroblasts when overexpressed is at the conjunction phrase because of the incitement Hey- Ras(17). 17Often were seen the malignancies in the various different people in the overexpressionD2 affixture, cyclin, for example T- cell lymphoma, testicular cancer, with the ovary's tumor.(2,33,39)23339The report card overexpressed that data present to perform that 1.1 kbs here changes to secret code a the C in the Graffi tumors- sliced the head to carry the cyclinD2 protein in the bitter end. The report card may not too be the information of the aberration, nontranslated, from the protein hereafter that were sliced the head to carry in the anticipant piece 17 kDas clearly discover in the tumorGris1 Were arranges agained and expresses in the normals the report card's gauze 1723339 织 of 1.1 kbs of 品 .Therefore, it is the conceivability's new isoform and also the possible in the light of ～ use the proto- oncogene standing alone of or use the full-length isoform in the cooperation aspect or because of others oncogenes.
D- The type cyclin expresses and during the period of change by the mitogens to respond G1- Cell period of to- The S is period(24,25,32). These respond D-The type cyclins becomes the synthesis because of the cyclin- Dependent kinases ( Cdk-4 is with the Cdk-6) through that highly kept the cyclin boxed(3,23, 27). Is°which arrive proof the phosphorylate pRb, and suggest a role recently For the rule of that synthesis at G all0- arrive- G1 Period The change is with rule that grow in the cell in the cell Circulation go forward(5,9,26). The cyclin D2 new isoform identifies Is a C here- In the protein that the bitter end sliced the head to carry to lack 133 contain what ammonia 基 Long isoform of sour, include the 23 kinds the last to contain the ammonia 基 sour From the cyclin box, but take 20 kinds contain the ammonia 基 sour and new to stretch. Whether cyclin D2 that we not and still know is sliced the head to carry or not is still retention Its bind the Cdks ability.However, the short isoform present Preface row is in this district of have to grow the aegis of the isoform 20 The 残 stays the thing in the 残 to stay the thing the L143, L146, P155 with the H156, only at Cyclin at an end box, suggest these 残s to stay the thing can There is the importance's function role, perhaps in the formation aspect There is Cdks a synthesis or because of other hand over with each other function partnership. Recent of proof to Cdk-The neutral sends to point out the making of D- type the activity Cyclins. Really, generally show the mutual influence of cyclin D2 because of DMP1, have to resemble the similar repetition protein of Myb, through its carboxyl- computer terminal Three of half( the 残 stay the thing 142 to 253) and that is not cyclin of D/ Cdk/ DMP1 The synthesis can be discovered(13). Because the DMP1 can't affect enough and each other, later on does not shelter the district from it Importance of for bind.
Show such as the analysis of our northern stain, the report card of 6.5 kbs Cyclin D2 the gene expresses in the high level quilts at respond the virus Tumor, not have at thoseGris1 The quilt is to arrange again, with At a thin 纱织 of nontransformed the 品 's variety. More and more limit Style expression that 1.1 kbs that report card instructions that Wering sliced to go to the head of the cyclin to carry can play a role in the development Thin 纱织 of specific medicine the brain that 品 resemble is a big part with the ovary, it Often expresses. D2 gene( because of both all that 6.5- And the report card of 1.1 kbs disrupt) Propagating of granulosa for emerging the weakness in that ovary Cell is with the granule with the stellate cerebellum interneurons (33,14). Report card, it think deeply to be sliced the head the cyclin that carry in the come-on The D2 can also have a role in these cells to type.u!:|s*d, http://www.100md.com
The cyclin family other member has the quilt to show two 择 a the ground have The coalescence report card predicts to slice the head to carry the protein. Such report card Have in the tumor cell the capable cyclin E is reported(18,28) And because of in the cyclin in various normals the D1 line up with tumor cell a line (4). however, two report card that 择 a the ground join together to describe Because the cyclinD1 and ～ is different from is discovers for cyclin D2 of those, When they change to secret code a the protein slice the head to carry in the little tiny part C-Computer terminal, be over.In the affixture,1 2 择 a ground coalescence The report card cheats to a to compare the isoform of the shorter cyclin C because everything go to the head to carry The cyclin box also is reported(22). Compare the shorter cyclin isoforms the energy to play the important role of the cell rule, At least in some specific medicines thin 纱织品 . Function of to the point Were sliced to go to the head of the cyclin D2 to carry to support to were appeared by that two 择 a ground Join togethered the information to cheat to for slicing go to the head the database of cyclin D2 at ET that carry the inside Because person is with the representative special features quilt Taieb with Jessus(38) atX. laevis.
We do not know to be sliced the head the isoform function role that carry In the normal cyclin in the cell D2, but we are in the inquisition its expression Pass by the cell period with particular hand over with each other function partnership Cloak-and-dagger protein.8^3%7s[, 百拇医药
This work drive the bestowal of Cancer from nation 007072 support Academic association Canada with award by the academic association from Canada 37994 Health research. The C.D. is what Cancer study to accept Social company studentship is with the FCAR Ph.D. Scholarship.8^3%7s[, 百拇医药
Adamson, M. C., J. 镀 is with the silver, with the C. A. Kozak. 1991. ThatCatch the rat gibbon ape leukemia the virus to suffer the container equal or the homology's thing: The 映 that inherit the gene shoots to act with container of suffering of a possibility in the rat. 滤 is over the sex microbiology183:778-781.8^3%7s[, 百拇医药
Bartkova,J.,E. Rajpert- De Meyts, N. E. Skakkebaek is with the J. 1999. At the adult mankind testis with the D in the testicular- Type cyclins Cancer: Relate to the cell type, and propagate, distinct, with Malignancy. J. Pathol. 187:573-581.
Die down, S., L. Bonetta, D. MacAllan, D. Fend off, A. Holder, C. 1994. The CDK6( PLSTIRE) is on using the cyclin consociation cyclin with the CDK4( PSK- J3)- Counting on that kinases that clear son gather D1. Oncogene9:71-79.'!2*{, http://www.100md.com
Betticher, D. C., N. 茸 , H. J. Altermatt, P. Hoban, W. 1995. The agent coalescence livings the product a novel Cyclin D1 report card. Oncogene11:1005-1011.'!2*{, http://www.100md.com
Datar, S. A., H. W. man, A. F. de la Cruz, C. F. Lehner, And B. A. Edgar. 2000. That果蝇 cyclin D- Cdk4 synthesis Promote the cell's growth. EMBO J. 19:4543-4554.'!2*{, http://www.100md.com
Denicourt, C., E. Edouard, with the E. Rassart. 1999. Oncogene In the Graffi rat the section's leukemia virus proviral myeloid leukemia making of inside activity Integrate. J. Virol. 73:4439-4442.'!2*{, http://www.100md.com
Deer of idle woman, S. F., P. W. 雌 , K. Louie, S. I. 芦苇 , A. 阿诺 , And R. A. Weinberg. 1993. Retinoblastoma of physically hand over with each other function Someone or other D cyclins protein. Cell73:499-511.'!2*{, http://www.100md.com
Ewen, M. E., H. K. Sluss, C. J. Sherr, H. Matsushime, J. 1993. The hand overing of function is with each other function that There is the mandible's D- The retinoblastoma protein of the type cyclins. Cell73:487-497.
Ezhevsky, S. A., A. Ho, M. Nod to signal hint- Hapak, P. K. Davis is with the S. 2001. Retinoblastoma tumor difference rule In the curb in the vivo G(1) cyclin- The counting on's kinase synthesis protein. 摩尔 . 21:4773-4784.w|, 百拇医药
Graffi, a. 1957. The rat's Chloroleukemia. Peaceful. N. Y. Acad. Sci. 68:540-558.w|, 百拇医药
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