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in situ isolation of mRNA from individual plant cells creation of cell-specific cDNA libraries

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LibrariesIn situ Isolation of mRNA from Individual Plant Cells: Creation of Cell-Specific cDNA

Gilchrist, and D Alexander EE Karrer, JE Lincoln, S Hogenhout, AB Bennett, RM Bostock, B Martineau, WJ Lucas, DG

doi:10.1073/pnas.92.9.3814 1995;92;3814-3818 PNAS

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Proc.Natl.Acad.Sci.USAVol.92,pp.3814-3818,April1995Biochemistry

InsituisolationofmRNAfromindividualplantcells:Creationof

cell-specificcDNAlibraries

(PCR/MonteCarloeffect/epidermalcell/guardcell/single-cellaspiration)

ERIKE.KARRER*t,JAMESE.LINCOLN*,SASKIAHOGENHOUTt,ALANB.BENNETT*§,RICHARDM.BOSTOCK*¶,

BELINDAMARTINEAU*11,WILLIAMJ.LUCAS**,DAVIDG.GILCHRIST*¶,ANDDANNYALEXANDERIItt

*CenterforEngineeringPlantsforResistanceAgainstPathogens,Davis,CA95616;tDepartmentofGenetics,VrijeUniversity,DeBoelelaan1087,1081HVAmsterdam,TheNetherlands;Departmentsof§VegetableCrops,1PlantPathology,and**Botany,UniversityofCalifornia,Davis,CA95616;andICalgeneInc.,Davis,CA95616

CommunicatedbyGeorgeBruening,UniversityofCalifornia,Davis,CA,December29,1994

ABSTRACTAmethodforisolatingandcloningmRNA

populationsfromindividualcellsinliving,intactplanttissues

isdescribed.Thecontentsofindividualcellswereaspirated

intomicropipettetipsfilledwithRNAextractionbuffer.The

mRNAfromthesecellswaspurifiedbybindingtooligo(dT)-

linkedmagneticbeadsandamplifiedonthebeadsusing

reversetranscriptionandPCR.Thecell-specificnatureofthe

isolatedmRNAwasverifiedbycreatingcDNAlibrariesfrom

individualtomatoleafepidermalandguardcellmRNAprep-

arations.Intestingthereproducibilityofthemethod,we

discoveredaninherentlimitationofPCRamplificationfrom

smallamountsofanycomplextemplate.Thisphenomenon,

whichwehavetermedthe"MonteCarlo"effect,iscreatedby

smallandrandomdifferencesinamplificationefficiency

betweenindividualtemplatesinanamplifyingcDNApopula-

tion.TheMonteCarloeffectisdependentupontemplate

concentration:thelowertheabundanceofanytemplate,the

lesslikelyitstrueabundancewillbereflectedintheamplified

library.QuantitativeassessmentoftheMonteCarloeffect

revealedthatonlyraremRNAs(<0.04%ofpolyadenylylated

mRNA)exhibitedsignificantvariationinamplificationatthe

single-celllevel.ThecDNAcloningapproachwedescribe

shouldbeusefulforabroadrangeofcell-specificbiological

applications.

ThecapabilityforisolatingandcloningmRNApopulations

fromindividualcellsinintact,livingtissueswouldallowthe

characterizationofadditionalgenesinvolvedincell-specific

biologicalprocesses.Individual-cellanalysisiscriticalwhen

eventsarelocalizedtemporallytooneorafewcells,suchasisdevelopment(1),signaltransduction(2),anddisease(3,4).

Suchanalysiscurrentlyisimpossibleusingconventional

mRNAisolationandcDNAcloningtechniques,sincetheylack

therequiredselectivityandsensitivity.

ExistingmethodsfortheanalysisofmRNApopulationsatthelevelofindividualcellscanmonitoronlyspecificgeneexpressionevents.Thesemethodsincludeinsituhybridization

withlabeledantisenseprobes(5)andPCRamplificationwith

labelednucleotides(6).Althoughtheseapproachesareveryeffectivetoolstostudycell-specificgeneexpressioninintact

tissues,theyhaveinherentlimitations.Onedrawbackisthat

thetissueiskilledpriortoorduringtheanalysis,thus

precludingtemporalmeasurementofgeneexpressionchanges

duringabiologicalevent.Anotherdisadvantageisthatonly

mRNAsofknownsequencecanbeanalyzed,sinceprior

knowledgeofthegeneisrequiredtodesignhybridization

probesorspecificPCRprimers.Thisrequirementmakesit

impossibletostudyunknowngenesthatareexpressedonly

transientlyorinspecificcelltypes.

Thepublicationcostsofthisarticleweredefrayedinpartbypagechargepayment.Thisarticlemustthereforebeherebymarked"advertisement"inaccordancewith18U.S.C.§1734solelytoindicatethisfact.Toovercometheseproblems,wehavedevelopedamethod

toisolateandclonecompletemRNApopulationsfromindi-

vidualcellsinsitu.Usingaprocedurewehavetermed"single-

cellaspiration,"mRNAwasisolatedfromindividualtomato

leafepidermalandguardcells.Amagneticbead-basedmethod

ofcDNAsynthesis,coupledwithPCRamplification,wasemployedtogeneratecDNAlibrariesfromsingle-cellamounts

ofmRNA.Intestingthemethod,aninherentlimitationtothe

PCRamplificationoflimitedamountsofcomplextemplate

wasdiscovered.Thenatureofthisphenomenon,herein

termedthe"MonteCarlo"effect,isdiscussedalongwithits

implications.

MATERIALSANDMETHODS

PlantMaterial.Tomatoseedsweresurface-sterilizedwith

合集下载

Development of Rapid One-Step Immunochromatographic Assay

Development of Rapid One-Step Immunochromatographic Assay

DevelopmentofRapidOne-Step

ImmunochromatographicAssay

Se-HwanPaek,*,1Seung-HwaLee,†Joung-HwanCho,*

andYoung-SangKim†

*GraduateSchoolofBiotechnologyand†DepartmentofChemistry,KoreaUniversity,5-ka,Anam-dong,Sungbuk-ku,Seoul136-701,Korea

Ananalyticalsystemforaone-stepimmunoassayhasbeenconstructedusingtheconceptofimmunochromatography.Thesystememployedtwodifferentantibodiesthatbounddistinctepitopesofananalytemolecule:anantibodylabeledwithasignalgenerator(e.g.,colloidalgold),whichwasplacedinthedrystateatapredeterminedsiteonaglass-fibermembrane,andanotherantibodyimmobilizedonthesurfaceofanitrocellulosemem-brane.Threemembranes,onewiththetracer,onewithimmobi-lizedantibody,andacellulosemembraneastheabsorbentofmedium(inasequencefromthebottom),wereattachedtoaplasticfilmandcutintostrips.Aqueousmediumcontainingana-lyteabsorbedfromthebottomendoftheimmunostripdissolvedthelabeledantibody,andtheantigen–antibodybindingcomplexformedwastransportedintothenextnitrocellulosemembranebytheflowcausedbycapillaryaction.Thecomplexsubsequentlyreactedwiththeimmobilizedantibody,whichgeneratedasignalinproportiontotheanalyteconcentration.Theconvectivemasstransferoftheimmunoreactanttothebindingpartnerallowedtheassaytobeperformedwithnohandlingofreagents.Thereaction,however,wascarriedoutundernonequilibriumconditions,whichresultedindecreasedsensitivityascomparedwithassaysper-formedinanequilibriummode(e.g.,ELISA).Tominimizesuchsacrifice,majorfactorsthatcontrolsystemperformancewereidentifiedandthesystemwasthendevisedunderoptimalcondi-tions.©2000AcademicPress

mRNA

mRNA

・３４４・　

的功能可能与酵母细胞中不尽完全相同。总之，ＣＢＣ　

蛋白在翻译过程中介导ＰＴＣ—ｍＲＮＡ的ＮＭＤ降解　

确定无疑。　

４小结　

ＰＴＣ—ｍＲＮＡ的ＮＭＤ降解过程如图１所示。在　

前体ｍＲＮＡ的剪接过程中，多种ｈｎＲＮＰｓ及剪接　

因子相互作用，剪接完成后于每个ＥＥＪ上游约２０　个核苷酸处形成一个ＥＪＣ。此时的ＥＪＣ可以称为　

“前ＥＪＣ复合体”，其５种组成蛋白中的ＤＥＫ和　ＳＲｍｌ６０为剪接因子，ＲＥＦ／Ａｌｙ为ｍＲＮＡ转运因　

子，而ＲＮＰＳ１和Ｙ１４结合ｈＵｐｆ３协同成熟ｍＲ—　ＮＡ，在ＲＥＦ／Ａｌｙ及ＴＡＰ／ｐｌ５等转运因子与核孔　

复合物的作用下转运出核。此后，ＥＪＣ复合物上部分　

因子随之脱落，细胞质中的ｈＵｐｆ２和ｈＵｐｆｌ通过与　ｈＵｐｆ３、ＲＮＰＳ１和Ｙ１４相互作用结合上来，形成“后　

ＥＪＣ复合体”。随后由帽结合蛋白ＣＢＣ起始第一轮　翻译，正常情况下，核糖体沿ｍＲＮＡ滑动将所有　

ＥＪＣ复合体逐个驱除，在到达最后一个外显子上的　

正常终止密码子处终止翻译，ＣＢＣ也在此过程中被　ｅｌＦ４Ｅ替换起始新一轮翻译。而在ＰＴＣ—ｍＲＮＡ分　

子中，核糖体在某个ＥＥＪ上游约５０～５５个核苷酸　

处遇到异常终止密码子，翻译停止，未脱落的后ＥＪＣ　复合体上的ｈＵｐｆ２、ｈＵｐｆｌ因子与翻译终止因子　

ｅＲＦ１、ｅＲＦ３相互作用，最终导致ＣＢＣ脱落，５　端没　有失去保护的ＰＴＣ—ｍＲＮＡ被迅速降解。　

ＮＭＤ降解机制广泛存在于各种含有内含子的　真核基因表达过程。据估计，约有５￣／４的人类基因没　

有内含子，其中主要一类是多拷贝的组蛋白和热休　

克蛋白基因，这类基因的表达水平较高，某个拷贝突　国外医学分子生物学分册２００２年第２４卷第６期　

变导致产生少部分Ｃ端缺陷截短蛋白并不十分有　

害，因而进化上对此类基因ｍＲＮＡ监测机制的选择　压力也大大降低［】　。而某些重要的单拷贝、无内含　

子基因（如ＭＣ４一Ｒ，其无义突变与遗传性肥胖有　

植生复习题(英文)

植生复习题(英文)

英文版植物生物学习题集（一）

Introduction, Cell Structure Part A

1. Plant life constitutes（组成）98% of the total biomass of the earth.

A: True B: False

正确答案是A

2. Having formed during the twentieth century, plant anatomy（解剖学）and plant physiology are the

most recently developed areas of botanical（植物学的）study.

A: True B: False

正确答案是B

3. In order to survive beyond the twenty-first century, humans will need to

A: recycle（使再循环, 反复应用）on a large scale（比例,数值范围）,

B: stop the increase in human population.

C: practice water and energy conservation（守恒）.

D: all of the above.

正确答案是D

4. Which of the following is NOT a feature（特征，起重要作用）of the scientific method?

A: hypothesis（假设）B: conjecture（推测,猜想）C: controls D: data collection

正确答案是B

5. The study of plant function is called

A: taxonomy.（分类学）B: physiology.（生理学）C: anatomy.（解剖学）D: morphology.（形态学）

研究生入学考试生物化学(RNA的加工与合成)历年真题试卷汇编3

研究生入学考试生物化学(RNA的加工与合成)历年真题试卷汇编3

研究生入学考试生物化学（RNA的加工与合成）历年真题试卷汇编3

(总分：78.00，做题时间：90分钟)

一、 判断题请判断下列各题正误。(总题数：10，分数：20.00)

1.(南开大学2009年考研试题)分解代谢激活蛋白(CAP)为同亚基二聚体，有2个cAMP结合位点。

（分数：2.00）

A.正确 √

B.错误

解析：

2.(南开大学2009年考研试题)几乎所有真核生物结构基因下游都存在polydT作为其转录产物polyA的模板

（分数：2.00）

A.正确

B.错误 √

解析：解析：真核生物polyA不为基因编码，由polyA聚合酶合成，不依赖模板。

3.(南开大学2008年考研试题)“RNA编辑”不改变mRNA的序列。

（分数：2.00）

A.正确

B.错误 √

解析：解析：“RNA编辑”是在初级转录物上增加、删除或取代某些核苷酸而改变遗传信息的过程。会改变mRNA的序列。

4.(南开大学2008年考研试题)一种乳糖操纵子的无代价诱导物叫做IPTG。

（分数：2.00）

A.正确 √

B.错误

解析：

5.(哈尔滨工业大学2008年考研试题)DNA的大部分序列被转录为mRNA，因而mRNA在细胞中的含量高于rRNA和tRNA。

（分数：2.00）

A.正确

B.错误 √

解析：解析：虽然DNA的大部分序列被转录为mRNA，其种类多但数量少，且不稳定，所以含量并不高于rRNA和tRNA。

6.(山东大学2006年考研试题)RNA的合成和DNA的合成一样，在起始合成前需要有RNA引物参加。

（分数：2.00）

A.正确

B.错误 √

解析：解析：DNA的合成在起始合成前需要有RNA引物参加。而RNA合成在起始合成前不需要有RNA引物参加。

7.(山东大学2005年考研试题)在具备转录的条件下，DNA分子中的两条链在体内都可能被转录成RNA。

（分数：2.00）

A.正确