A NOVEL DNA-BASED MICROFLUORIMETRIC METHOD TO EVALUATE ANTIMALARIAL DRUG ACTIVITY

Unit 3 Text A百分之七十的解决方案我喜欢练习跆拳道，其一招一式精准潇洒，犹如翩翩起舞。

不过我尤其醉心于过招时的兴奋。

步入拳坛，躲闪、侧避、脚踢、临空飞起霹雳腿。

我酷爱参加各种循环比赛时，在点到为止的格斗中肾上腺素飙升的激情。

我参加过全国比赛，在泛美运动会预赛中赢得铜牌。

这都是很久以前的事了。

随后发生了太多的事情：在医学院就读，完成了内科实习，生了一儿一女。

最后我成了带教全科医生，再后来又得了一种慢性病。

和众多自体免疫疾病患者一样，我的问题出现二十年后才确诊：体力和精力下降，平衡出现问题，可怕的脸部阵痛，视力下降。

出现足下垂后，我最终被确诊罹患多发性硬化症。

导致免疫细胞攻击大脑原因何在？我的医生指出，导致多发性硬化症的危险因素中，遗传因素只占 10%到 30%，其余是各种未知的环境因素。

他没有告诉我如何应对这些未知因素，仅仅开了降低复发的干扰素和共聚物-1。

他说复发越少，一来致残程度越底，二来再过十年，我还能如那时一样行走自如、工作有效、生活无妨，这种可能性会大增。

我立即开始药物注射。

随后四年只有一次复发：右臂短暂无力。

然而，我日趋虚弱，机能和活动耐力日益丧失。

我先是不能慢跑了，随后站立变得困难。

即便用了最新的药物治疗，最后步行和端坐也是累人之事，我要靠一张斜背轮椅。

随着时间流逝，因病卧床不起不可避免，这越来越明显。

我到了人生的十字路口。

不管是对我们理念的热衷，亦或是拒绝悄无声息地步入死亡，这些动力让医生亲身试验已经存在数百年了。

我只有两个选择：要么调节心态，接受现实，即虽然接受最佳治疗，但是残疾日剧，要么更为积极主动，自己应对健康状况。

我曾是个斗士，如今已身心疲惫。

不过，只要我能，我还想起身行走，即便区区几步。

我开始研究文献，一篇篇阅读 PubMed 上的文章，我心里明白当今临床治疗萌发于数年或数十年前的基础科学文献所撒的种子。

我希望能找到阻止我日益恶化残疾的魔弹。

一开始，我寻找多发性硬化症药物的最新动物实验文章。

生物多样性 2016, 24 (2): 237–243 doi: 10.17520/biods.2015205 Biodiversity Science http: //・综述・InDel标记的研究和应用进展杨洁赫佳王丹碧施恩杨文宇耿其芳王中生*(南京大学生命科学学院, 南京 210023)摘要: InDel是指在近缘种或同一物种不同个体之间基因组同一位点的序列发生不同大小核苷酸片段的插入或缺失(insertion-deletion), 是同源序列比对产生空位(gap)的现象。

InDel在基因组中分布广泛、密度大、数目众多。

InDel 多态性分子标记是基于插入/缺失位点两侧的序列设计特异引物进行PCR扩增的标记, 其本质仍属于长度多态性标记, 可利用便捷的电泳平台进行分型。

InDel标记准确性高、稳定性好, 避免了由于特异性和复杂性导致的后续分析模糊。

此外, InDel标记能扩增混合DNA样品和高度降解的微量DNA样品, 并进行有效分型。

InDel标记目前已开始应用于动植物群体遗传分析、分子辅助育种以及人类法医遗传学、医学诊断等领域。

随着位于功能基因上InDel 标记的开发, 结合染色体步移和基因精细定位, 可将这些标记应用于相关物种经济性状的功能基因的筛选, 有利于优良基因的进一步开发和利用。

关键词:分子标记; InDel; SNP; SSRProgress in research and application of InDel markersJie Yang, Jia He, Danbi Wang, En Shi, Wenyu Yang, Qifang Geng, Zhongsheng Wang*School of Life Sciences, Nanjing University, Nanjing 210023Abstract: InDel indicates insertions or deletions (insertion-deletion) of nucleotide fragments of different siz-es at the same site in the genome sequence between the same or closely related species and is a gap in se-quence derived from alignment of the homologous sequence. InDel is widely distributed across the genome and occurs in a high density and large numbers in a genome. The InDel polymorphic molecular marker is a PCR-amplified marker that is based on specific primers designed from both sides of the site of sequence of insertion / deletion. It is essentially a length polymorphic marker still, and one can use the convenient elec-trophoresis platform for genotyping. InDel molecular markers have the advantage of high accuracy and good stability, which help to avoid confusion in subsequent analysis due to marker specificity and complexity, as is often seen in other length polymorphic markers. Furthermore, mixed or highly degraded DNA samples can be successfully amplified with InDel markers, and effectively typed. Because of its abundance, convenient typing platform and other advantages, InDel molecular markers have been applied to genetic analyses of an-imal and plant populations, molecular assisted crops and farmed animal breeding, human forensic genetics, medical diagnostics and other research areas. The development of the InDel molecular marker located on functional genes, combined with chromosome walking and fine gene mapping, has enabled the application of these molecular markers in the screening of genes related to important economic traits, which is conducive to the further development and utilization of these valuable genes. In this review, on the basis of an overview of the InDel marker development and applications, we discuss some of the technical limitations of the develop-ment and limited efficiency of genetic analysis, as well as potential future applications in the fine mapping and genetic structure of large numbers of individuals.Key words: molecular marker; InDel; SNP; SSR插入/缺失(insertion-deletion, InDel)是指在近缘种或同一物种不同个体之间基因组同一位点的序——————————————————收稿日期: 2015-07-15; 接受日期: 2015-12-17基金项目: 国家自然科学基金(31100270)∗通讯作者Author for correspondence. E-mail: wangzs@238 生物多样性 Biodiversity Science第24卷列发生了不同大小核苷酸片段的插入或缺失, 即一个序列上某一位点相比同源的另一个序列插入或缺失了一个或多个碱基(Weber et al, 2002)。

Chromosomal imbalance reportA novel de novo 1.8Mb microdeletion of 17q21.33associated with intellectual disability and dysmorphic featuresE.Preiksaitiene a ,*,K.Männik b ,V.Dirse a ,A.Utkus a ,Z.Ciuladaite a ,J.Kasnauskiene a ,A.Kurg b ,V.Ku c inskas aa Department of Human and Medical Genetics,Faculty of Medicine,Vilnius University,Vilnius,Lithuania bInstitute of Molecular and Cell Biology,University of Tartu,Tartu,Estoniaa r t i c l e i n f oArticle history:Received 31October 2011Accepted 13July 2012Available online xxx Keywords:17q21.33deletion Intellectual disabilitya b s t r a c tWe report on a de novo 17q21.33microdeletion,1.8Mb in size,detected in a patient with mild intellectual disability,growth retardation,poor weight gain,microcephaly,long face,large beaked nose,thick lower lip,micrognathia and other dysmorphic features.The deletion was detected by whole-genome geno-typing BeadChip assay and involves the genomic region between 45,682,246and 47,544,816bp on chromosome 17.Among the 24RefSeq genes included in this deletion are the CA10and CACNA1G genes that are involved in brain development and neurological processes.A possible candidate gene for the prenatal and postnatal growth retardation is the CHAD gene,which product chondroadherin is a cartilage protein with cell binding properties.These three genes may be responsible for the patient ’s phenotype.Ó2012Elsevier Masson SAS.All rights reserved.1.Method of detection 1.1.CytogeneticsCytogenetic analysis was performed on peripheral blood lymphocytes using a conventional G-banding technique at 400e 500band resolution.The chromosomal analysis of the patient revealed a normal male karyotype:46,XY.1.2.SNP-CGHSubmicroscopic chromosomal alterations were screened by In finium HD whole-genome genotyping assay with the HumanCytoSNP-12BeadChip (Illumina Inc.,San Diego,CA,USA),which covers the entire genome with an average spacing of 9.6kb and allowing an average resolution of 31kb.The sample was pro-cessed and the assay was performed according to a routine protocol provided by the manufacturer.Genotypes were called by GenomeStudio GT module version 1.7(Illumina Inc.),and further analysed with QuantiSNP version 1.1[1]and KaryoStudio version 1.0.3softwares (Illumina Inc.).1.3.Chromosomal anomalyWhole genome genotyping analysis of the patient revealed a deletion in chromosome band 17q21.33(Fig.1).The telomeric breakpoint was located within a 13,060bp interval delimited by two probes located in the intron 8of the CA10gene:probe rs1393728was deleted whereas probe rs7504022was not deleted.The centromeric breakpoint was located between probe rs2586469(deleted)and probe rs8082154(not deleted)within a 935bp region.The genomic position of the deletion was between 45,682,246bp and 47,544,816bp (Build 36:Mar.2006).The minimal and maximal size of the deleted region was 1,862,570bp and 1,876,565bp,respectively.1.4.Method of con firmationFISH using BAC clone RP11-121F10(45,949,240e 46,110,829)mapping to 17q21.33region (CloneCentralT human BAC Clone Locator)was performed on metaphase chromosomes of the patient and his parents.1.5.Causative of the phenotypeThe analysis of both parents by FISH did not reveal the deletion thus indicating that the 17q21.33deletion in the patient was de*Corresponding author.E-mail address:***************************(E.Preiksaitiene).Contents lists available at SciVerse ScienceDirectEuropean Journal of Medical Geneticsjournal ho mep age:htt p://www.elsevier.co m/locate/ejmg1769-7212/$e see front matter Ó2012Elsevier Masson SAS.All rights reserved./10.1016/j.ejmg.2012.07.008European Journal of Medical Genetics xxx (2012)1e 4novo .The deletion is not reported in the Database of Genomic Variants (http://projects.tcag.ca/variation/)and involves several genes which are implicated in brain development and neurological processes.2.Clinical descriptionThe patient was a 17years old male,the first child of healthy non-consanguineous Lithuanian parents aged 29(mother)and 25years (father)at birth of the proband.The proband had an older healthy maternal half-brother.He was born after an uneventful pregnancy and a normal delivery at 39weeks of gestation.His birth weight was 2550g (<3rd centile)and length 51cm (25th percen-tile).He had a normal motor development,but his language development was severely delayed.Growth retardation and poor weight gain were observed from early infancy.At the age of 10years the boy was evaluated for delayed growth by a paediatric endo-crinologist.His bone age was appropriate for his chronological age.At the age of 14years his height was 141cm (3rd centile),weight 27kg (<3rd centile)and head circumference 49cm (<3rd centile).He experienced recurrent respiratory tract infections and had dif ficulty with sleeping e restless,disturbed and inconsistent night-time sleep.With advancing age he became more prone to outbursts of anger and irritable.On examination at the age of 17years his height was 171cm (10e 25th centile)and weight 51kg (3rd centile)had normalized,while he still had a microcephaly (52cm,<3rd centile).Other features were a long face,large beaked nose,thick lower lip,micrognathia,malocclusion of the teeth (Fig.2),clino-dactyly of the 5th finger and a gap between the first and second toe.His pubertal development was considered to be normal.Refractive errors (myopia)were diagnosed by an ophthalmologist.A psycho-logical evaluation at the age of 16years yielded a verbal score of 68,a performance score of 63and a total IQ score of 65on the Wechsler Intelligence Scale for Children (WISC-III).3.DiscussionWe present the clinical and molecular characterization of a de novo 17q21.33microdeletion,1.8Mb in size,detected in a patient with mild intellectual disability and dysmorphic features.ThisisFig.1.1.8Mb deletion in chromosome band 17q21.33,between 45,682,246bp and 47,544,816bp (Build 36:Mar.2006)detected using In finium HD whole-genome genotyping assay with the HumanCytoSNP-12BeadChip is presented in the bottom.Above the map of deleted (in black lines)and duplicated (in white lines)regions according DECIPHER,ECARUCA and previously reported cases in literature ispresented.Fig.2.Front and side lateral views of the patient.Note long face,thick lower lip (A),large beaked nose and micrognathia (B).E.Preiksaitiene et al./European Journal of Medical Genetics xxx (2012)1e 42thefirst report of a17q21.33microdeletion and we found no publications with overlapping submicroscopic deletions.A search for further patients revealed three individuals with informative overlapping deletions through the DECIPHER(http://decipher. )and ECARUCA()databases(Fig.1, Table1).Two duplications overlapping the17q21.33region are reported in the literature[2,3],suggesting the presence of dosage sensitive genes that are critical for neurodevelopmental processes in this gene-rich chromosomal region.The patient reported in the DECIPHER database(ID878)with a17q21.33q22microdeletion, 5.8Mb in size,has intellectual disability and few other clinical features in common with our patient.His phenotype at the age of4years and10months was remarkable for a relatively low weight(25th centile)and small head circumference(3rd centile)compared to his height(75th centile).He also has a brachycephaly,small chin,a scalp defect (aplasia cutis congenita),round face,relatively large andfleshy ears, partial cutaneous syndactyly between second and third toes and abnormalflexion of the thumb(personal communication). Although larger in size,this deletion provides useful information about17q21.33related deletions and the presence of common clinical features allows greater certainty of the pathogenic nature of the rearrangement found in our patient.The second patient reported in the DECIPHER database(patient ID255632)had an overlapping deletion,1.92Mb in size,spanning the proximal two-thirds of the chromosomal alteration in our patient.Both patients shared intellectual disability and dysmorphic features,but the latter were not detailed in the ID255632and thus could not serve for identification of common clinical features.The other symptom described in this individual was an oesophageal atresia/stenosis,which was not observed in our patient.Interestingly,tracheoesophagealfistula was also characteristic for the17q21.3q23deletion reported in the ECARUCA database. Probably the critical gene for this clinical feature is not involved in our deletion.Intellectual disability and several minor anomalies are shared by our patient and the patient reported in the ECARUCA database:low birth-weight,short stature,microcephaly,and micrognathia.However,this deletion is not characterized by genome-wide array and the size of the rearrangement is too large to allow useful genotype phenotype correlations.The potential clinical relevance of the chromosomal alteration in our patient is supported by its gene content.The deletion encom-passes up to24known genes,21protein coding genes and3non-coding RNA genes(Human NCBI36Assembly,hg18).Seventeen of the known genes have been shown to be expressed in brain(CAC-NA1G,WFIKKN2,EME1,ANKRD40,LUC7L3,RSAD1,CA10, NME1e NME2,MRPL27,SPAG9,LRRC59,ACSF2,UTP18,TOB1,MYCB-PAP,MBTD1and EPN3).Therefore these genes are functional candidates contributing to the impairment of the central nervous system in our patient.However,data that allow a clinical evaluation of the presumptive haploinsufficiency effect are available for just a few of these deleted genes.Among them is the CACNA1G gene encoding a voltage-dependent calcium channel which is thought to be involved in neuronal oscillations and resonance,in pacemaking activity in central neurons,as well as in neurotransmission. Therefore its haploinsufficiency might cause disturbance in neu-rodevelopmental processes.CACNA1G gene mutations have not been reported in humans.However,in recent studies its association with autism[4,5]and epilepsy[6]has been implicated.These studies support that this gene as a good candidate gene for intel-lectual disability.The other gene which haploinsuficiency may be related to our patient’s impaired cognitive functioning is the CA10 gene.CA10is highly expressed in the human brain(cerebellum, frontal cortex,parietal cortex,midbrain),testis,salivary glands,and kidney[7,8],and it is thought to play a significant role in the central nervous system,especially in brain development.A possible candidate gene for the prenatal and postnatal growth retardation may be the CHAD gene,which product chondroadherin is a cartilage protein with cell binding properties.The expression of chondroadherin was studied in rat tissues and during postnatal femoral head development.The high expression of this cell binding protein in a dynamic region of cartilage suggests an important role for chondroadherin in the regulation of chondrocyte growth and proliferation[9].Tasheva et al.(2004)[10]provided novel infor-mation about the broad expression of Chad in many tissues, including cornea,retina,Purkinje cells of the cerebellum,periph-eral nerves,blood vessels of various tissues,pancreatic islets,and ovary.In conclusion,we report on a novel de novo17q21.33micro-deletion in a patient with intellectual disability and dysmorphic features.This observation supports the role of CA10and CACNA1G genes in cognitive development.Detailed clinical examination of additional patients with a similar microdeletion is needed for a complete description of the phenotype and for the elucidation of the influences of CA10,CACNA1G and CHAD haploinsufficiency on that phenotype.Table1The main clinical features of17q21.33related chromosomal alterations.17q21.33RelatedanomaliesSize Main clinical phenotype ReferencesDel17q21.33 1.8Mb Mild mental retardation,growth retardation,poor weight gain,microcephaly,long face,large beaked nose,thick lower lip,micrognathia,malocclusion of teeth,clinodactyly ofthe5thfingers,gap betweenfirst and second toe,myopiaOur patientDel17q21.33q22 5.8Mb Mental retardation,poor weight gain,normal height,microcephaly,brachycephaly,round face,micrognathia,fleshy and relatively large ears,aplasia cutis congenita,partialcutaneous syndactyly of2e3toes,abnormalflexion of the thumbDecipher878Del17q21.33 1.9Mb Mental retardation,general abnormalities of face,oesophageal atresia/stenosis Decipher255632 Del17q21.3q23Mental retardation,tracheo e oesophagealfistula,club foot varus,low birth-weight,shortstature,brachycephaly,plagiocephaly,microcephaly,dystopia canthorum,hypertelorism,short palpebralfissures,palpebralfissures slant up,round face,micrognathia,downturnedcorners of the mouth,long philtrum,cleft uvula,lose skin in neck,synostosis offingers,proximal placement of thumbEcarucaDup17q21.33 1.1Mb Mental retardation,microcephaly,deafness,vision abnormalities,epicanthic folds,palpebralfissures slant up,short palpebralfissures,high palate,auricular tags,clinodactyly,pes cavus,syndactyly2e3of toes,taperingfingersZahir et al.,2009Dup17q21.3q22Palpebralfissures slant up,colobomata,large beaked nose,prominent nasal tip,microghathia,wide-spaced nipples,syndactyly,hypoplastic nails,coronal hypospadias Leana-Cox et al.,1993E.Preiksaitiene et al./European Journal of Medical Genetics xxx(2012)1e43AcknowledgementsWe would like to thank Dr.Björn Menten for the clinical infor-mation of the patient with17q21.33q22microdeletion.The research leading to these results has received funding from Euro-pean Union’s Seventh Framework Programme[FP7/2007-2013] under grant agreement no.223692CHERISH project.References[1]S.Colella, C.Yau,J.M.Taylor,G.Mirza,H.Butler,et al.,QuantiSNP:anObjective Bayes Hidden-Markov Model to detect and accurately map copy number variation using SNP genotyping data,Nucleic Acids Res.35(2007) 2013e2025.[2] F.R.Zahir,nglois,K.Gall,P.Eydoux,M.A.Marra,J.M.Friedman,A novel denovo1.1Mb duplication of17q21.33associated with cognitive impairment and other anomalies,Am.J.Med.Genet.A149(2009)1257e1262.[3]J.Leana-Cox,S.Levin,R.Surana,E.Wulfsberg,C.L.Keene,L.J.Raffel,B.Sullivan,S.Schwartz,Characterization of de novo duplications in eight patients by usingfluorescence in situ hybridization with chromosome-specific DNA libraries, Am.J.Hum.Genet.52(1993)1067e1073.[4]S.P.Strom,J.L.Stone,J.R.Ten Bosch,B.Merriman,R.M.Cantor,et al.,High-density SNP association study of the17q21chromosomal region linked to autism identifies CACNA1G as a novel candidate gene,Mol.Psychiatry15 (2010)996e1005.[5]J.Krey,R.Dolmetsc,Molecular mechanisms of autism:a possible role for Ca2þsignaling,Curr.Opin.Neurobiol.17(2007)112e119.[6] B.Singh, A.Monteil,I.Bidaud,Y.Sugimoto,T.Suzuki,et al.,Mutationalanalysis of CACNA1G in idiopathic generalized epilepsy,Hum.Mutat.28 (2007)524e525.[7]N.Okamoto,K.Fujikawa-Adachi,I.Nishimori,K.Taniuchi,S.Onishi,cDNAsequence of human carbonic anhydrase-related protein,CA-RP X:mRNA expressions of CA-RP X and XI in human brain,Biochim.Biophys.Acta1518 (2001)311e316.[8] A.Aspatwar,M.E.Tolvanen,S.Parkkila,Phylogeny and expression of carbonicanhydrase-related proteins,BMC Mol.Biol.31(2010)11e25.[9]Z.Shen,S.Gantcheva, B.Mânsson, D.Heinegârd,Y.Sommarin,Chon-droadherin expression changes in skeletal development,Biochem.J.330 (1998)549e557.[10] E.S.Tasheva,A.Ke,G.W.Conrad,Analysis of the expression of chondroadherinin mouse ocular and non-ocular tissues,Mol.Vis.10(2004)544e554.E.Preiksaitiene et al./European Journal of Medical Genetics xxx(2012)1e4 4。

中山大学细胞生物学英文题库1. You think you might have discovered a new organelle using electron electron microscopy, but you are not sure if it's real or an artifact of your technique. What might you do to prove it is real?a. Look for the structure in many similarly prepared specimens from the same source.b. Look for the structure in many similar prepared specimens from different sources.c. Look for the same structure in specimens prepared using different staining protocols.d. Look for the same structure in specimens prepared using different (or no) fixatives.2. Traditional genetic approaches to problems of cell biology have followed the path of information from gene to protein. Biochemical approaches have traced proteins back lto the DNA that encodes them, i.e., from protein to gene. This chapter has given you the basics to follow either path. Assume you are studying a problem relating to cell function. Briefly outline the basic steps you would take if you were starting with a gene and trying to deduce the function of the protein. Now outline the steps that you would take to get from a specific protein to a sequenced, isolated gene.1. Indicate whether the following statement is true or false. If it is false, expolain why in Nuclease-chypersensitive sites in chromation are located in the linker DNA between nucleosomes.(F)2. Short answera. Evolutionary conservation of histone proteins.b. nuclear matrix3. Suppose you discover a temperature-sensitive nutant whose nucleus failed to accumulate certain nuclear protein atan elevated (restrictive) temperature, but continued to accumulate other species. What conclusions might you draw about nuclear localization and the nature of this mutation?Cells possess more than one NLS. receptor that recongnize different localization sequences, and that the mutation was occuring in a gene that encoded one of these NLS receptor species.1. Fill in the blanks.A functional chromosome regrires the DNA sequence element at least one_______ to permit the chromosome to copied one to facilitate proper segregation of its two copies at mitosis and two ________ to allow the chromosome to be maintianed betwiin cell generations.(ARS; CEN)2. Which statemint best characterizes the nuclear localization signal?a. The NLS is tupically a small molecular weight metabolic intermediate.b. The NLS is a shetch of hydrophobic amino acids on a proteins N terminus.c. The NLS is one or two streches of basic amino acids on a protein'sC-terminus.d. The NLS is a steroid that binds to DNA.(C)3. You suspect that a new hormone you are testing functions to stimulate myosin synthesis by acting at the transcriptioned level. What type of experimental evidence would support this contention.1. Indicate whether the following statement is ture or false. If a statement is false, exlain why.a. Both myosin-! and myosin-!! have a rollike tial that allows them to polymerize into bipolar filaments, which is crucial for moving groups of oppositely oriented actin filaments past each other.b. Sarcomere shortening is caused by the myosin filaments sliding past the actin filaments with no charge in the length of either type of filaments.2. If you treated cells with a drug that interferes with microtubuls such as colchrarne, which of the following would result?a. Cell shape would be disruptedb. Mitosis and merosis would not occurc. The intracellular location of organelles would be disruptedd. All of the above would result3. Some antibodies capable of binding to kinesin are able to block organelle movement, whereas other anti-kinesin antibodies seem to have bittle effect: How can you explain this obsenation?1.Would you agree with the statement that the centrosome plays a key role in determining that rates of lengthening and shortening of the microtubules of an animal cell? Why or why not?pare and contrast the following cytoplasmic dynein v.s. Kinesin3.Differences in microtubular stability and function between different cells (or different locations within a cell) may be due to which of the following?a. the expression of different tubulin isformsb. the number of protofilaments composing a single microtublec. the presence of different MAPsd. all of the above1.What type of antibody could you use to distinguish between isolated coated vesicles that had formed at the TGN from those that had formed at the plasma membrane?2.Which of the body would you probably be the richest in HDL receptors?a. stomach cellsb. liver cellc. muscle celld. epitholial cells(D)3.A certain integral protein of the plasma membrane has two transmembrane domains in wihich both the C-terminals and the N-terminals are directed toward the cell extenor in the mature protein. Where are the C-termini located when the proteinis newly translocated into the RER membrane? Into the Golgi? Into the vesicles?The nicotinic acetylcholine receptor is membrane-bound protein that also functions as a ligand-gated ion channel. Upon binding acetyleholine, the channel opens and sodi1. Fill in the blanks in the following statements:In cells treated with ______ the Golgi appearatus largely disappears and the Golgi proteins end up in the ER, where they intermix with ER proteins.2. Indicate whether the following is true or false. (T)If a statement is false, explain why.All of the glycoprotions and glycolipids in intracellular membranes have their oligosaccharides facing the luminal side, whereas those in the plasma membrane have their oligosaccharides facing outside the cell.3. You have labeled the lipids on a patch of rough ER membrane with a fluorescent probe. After a few minutes, the probe shows up in the membranes of the cis Golgi. Now you treat the cells with the drug brefieldin A where might the fluoresent probe show up next?a. trans Golgi networkb. endoplasmic reticulumc. plasma membraned. secretory vesicles1. Fill in the following blanks:The amino-terminal ER signal poptide of a soluble protein itself have two functions. In addition to directing the protein to the ER mambrane, it also serve as a ________.(起始跨膜信号)2. True of false: if false, explain why.In mammalian cells, the import of protein into ER begins before the polypeptide chain is synthesized, that is, it occures to co-translationality.3. Which of the following lack a signal sequence?a) acid hydrolase enzymes synthesized in macrophage cells.b) glycolytic enzymes synthesized in liver cells.c) polypeptide gormones synthesized in endocrine cells.d) antibody hormones synthesized in plasma cells.1.Which of the following explain why microsomes can't be seen viewed with eletron microscope?a) They are far too small.b) They are antifats of homogenization and centrifugation.c) They are transparent to electrons.d) They actually can be seen in election micrographics of cells.2.If you were to add a drug to interfere with the ability of ribosome to bind to mRNA, what effect would this be expected to have to the structure of the RER?3.Which, if any, of the following polypeptides would be expected to look, a signal peptide antibodies, acid phosphatase, hemoglobin, ribosomal protein, glycophorn.1.Would you expect the import of substances such as ADP or Pi to lead to a decrease in the proton-motive force? Why?Yes, because they occur at the expense of either the potential difference acress the membrane(in the case of the ATP/ADP exchange) or of the proton gradient (in the case of the uptake of Pi)2.Why would you expect the movement of protons into the intermembrane space to have an effect on the enzymes o fthe cytosol?Because the outer membrane is freely permeable to protons, therefore the protons concentration in the intermembrane space would rapidly epuilibrate with that of the cytosol, lowing the cytosolic pH.ing differential centrifugationm, you are trying to isolate peroxisomes and glyoxysomes from a mixture of cellular organelles. After a few centrufugation steps, you think you may have a relkatively pure suspension. How might you determine that your suspension does, indeed, have there organelles?a) Do an assay for the enzyme catalase.b) Do an assay for the enzyme succinate debydrogenase.c) Look in the microscope for a double -membrane bound organelle.d) Assay you suspension for nucleic acid.(A)1.Which of the following statements about the outer mitochondrial membrane is not true?a) The outer membrane is impermeable to hydrogen ions.b) The outer membrane is highly permeable to substances of small molecular weight.c) The outer membrane is about 50% lipid and 50% protein by weight.d) The outer membrane contains proins.2.Which of the follwing is not an electron carrier class in the electron transport chain?a) flavoproteinsb) cytochromesc) iron-sulfur proteind) cytochrome coxidase3.Short answer: Na-k ATPase from red blood cells can be made to synthesis ATP1. In most cases, RNA acts as an intermediary molecule between the genetic information and the final product of that information, proteins.What are the advantages of having an intermediary as opposed to translating the DNA directly into protein?2.The textbook states that the nucleotide sequence of splice sites is "ancient origin". How do we know this sequence is ancient?a. It is widespread and occurs in even the most primitive eukaryotes.b. The number of mutations that have accumulated in the sequence indicates that it must have been in cells for millions of years.c. It is only found in organisms that are highly evolved.d. It codes for ancient proteins.3. If cultured cells are incubated with 14^C-merhionine the radiolab will appear:a. in all the rRNAs simultaneously.b. first in the 45S RNA, followed by the 32S RNA, followed by the 28S RNA.c. first in the 45S RNA, followed by the 28S RNA, followed by the 5S RNA.d. first in the 28S RNA, followed by the 32S RNA, followed by the 45S RNA.pare and contrast the folowing:(1) "one gene-one enzyme" vs "one gene-one transcription unit".(2) "upstream promoters" vs "internal promoters"2.The signification of methylation of pre-rRNA is probablya. To ensure that the final rRNAwill assume the correct tertiary structre.b. To prevent the pre-rRNA from being processed.c. To prevent degredation of the parts destined to become mature rRNA.d. None of the above.3.The following complex of macromolecules is found in a cell: DNA attached to RNA polymerase, attached to a growing chain of RNA attached to a ribosome, attached to two tRNAs, attached to a growing chain of amino acids. The cell must be:a. A plant cell.b. An animal cell.c. A virus.d. A prokaryotic cell.4.The 5s rRNA differs from the other cellular rRNAs in which of the following ways?a. The genes are located outside the nucleolus.b. The genes are transcribed by RNA polymerase Ⅲ.c. The promoter region of the 5s rRNA gene is internal to the gene.d. All of the above.1. Ca^2+, IP3, and cAMP are very different second messagers. In what ways are their mechanisms of action similar? In what ways are they different?2. Suppose that epinephrine and nonepinephrine could initate a similar response in a particular target cell. How could you show that the two compounds act by binding to the same cell-suface-receptor?3. If you break liver cells b homogenization separate the broken cell membranes (particulate) from the cytoplasm (sohuble) and add epinephrine to the soluble portion, the result will bea. the production of cAMPb. binding of the epinephrine to its receptorc. activeation of adenylyl cyclased. none of the aboveA gene encoding growth hormone in rats was introduced into mice. The construst used consisted in an open reading frame encoding rat growth hormone fused to a mouse promoter A. the growth hormone sequence was obtained from a rat cDNA library the only available source of sequence information. Fertilized mouse egg cells were transformed with the rat growth hormone construst and expression of the rat growth hormone was detected. Transformation of fertilized egg cells with rat growth hormone cDNA alone did not result in expression.1. Why was it necessary to fuse the rat growth hormone sequence to a promoter to obtain expression?a. The rat sequence was obtained from a cDNA library and therefore did not include a promoter sequence.b. No factors that recognized the rat promoter sequence were present in the mouse.c. The rat promoter was contained with the rat sequence but was inactivated in the mouse.d. Rat growth hormone is inactivated in mice.2. When the eggs were placed under conditions conducive to activation of the mouse promoter one of the resulting off spring grew large than the expected size. The appearance of the large mouse is consistent with one the following statements:a. That mouse grew bigger because it produced more mouse growth hormone.b. That mouse grew bigger because it was more aggressive and ate more mouse chaw.c. That mouse grew bigger because the mouse promoter A was activated result in increased production of rat growth hormone.d. The mouse grew bigger because his nose was longer.1. Neleic acid hybridization can be used as a measure of evolutionary relationships between species of the following statement about the DNA of related species, which is truea. Closely related species from hybrid DNAs with relatively low melting temperatures.b. Closely related species from hybrid DNAs with relatively high melting temperatures.c. There is no correlation between DNA hybrid meltiong temperature and relatedness of species.d. One cannot creat DNA hybrid molecules from DNAs of closely related species. techniques you might use to answer each of the following questions.a. Does the DNA on the hair found at the scene of a crime match that of the defendant in a criminal trial?b. Where in a cell are the carbobydrates added to the glycoprotein hormones that are exported?1. Which type of the cell would you expect to achieve the largest volume: a highly flattened cell or a splherical cell? Why?2. All of the following individuals contributed to cell theory except:a. Robert Hookeb. Matthias Schleidenc. Theodor Schwannd. Rudolf Virchow3. Cell theory includes all of the following expecta. All organisms are composed of ome or more cells.b. The cell is the most primitive form of life.c. The cell is the structual unit of life.d. Cells arise by division of preexisting cells.4. Given that cell structure reflects cell function, what structual features would you predict in the following?a. Cell that line the digestive tract and take up nutrients across the wall of the intestine into the blood.b. Cells that synthesize and secrete protein such as an insulin producing cell from the pancreas or an antibody-producing lymphocyte.1. Fill in: A specific tripeptide, sequence, the _____________ is found in one of the repeats and is a central feature of the site responsible for cell binding by fibronectin.2. True or false:Although most types of collagen assemble into fibrilsl type**** collagen assembles into a shoetlike network that forms the core of all basal lamina.3. The addition of peptide within RGD sequence would probably ________ the binding of cultured cells to a fibronectin coated dish.a. enhanceb. inhibitc. have no effect ond. none of the above。

西南大学网络与继续教育学院[1194]《生活中的DNA科学》单项选择题1、朊粒病的共同特征中不包括（）1.潜伏期长，达数月、数年甚至数十年2.一旦发病呈慢性、进行性发展，以死亡告终3.表现为海绵状脑病或蛋白质脑病4.产生严重反应和免疫病理性损伤2、下面哪种酶是在重组DNA技术中不常用到的酶（）1.限制性核酸内切酶2.DNA聚合酶3.DNA连接酶4.DNA解链酶3、长期接触X射线的人群，后代遗传病发病率明显升高，主要原因是该人群生殖细胞发生（）1.基因重组2.基因突变3.基因互换4.基因分离4、抗VD佝偻病属于：（）1.常染色体显性遗传病2.常染色体隐性遗传病3.X连锁显性遗传病4.X连锁隐性遗传病5、法医DNA技术中的三大基本技术指（）①DNA 指纹技术②荧光显微技术③PCR 扩增片段长度多态性分析技术④线粒体DNA 测序1.①②③2.②③④3.①②④4.①③④6、相互连锁的两个基因位于（）上1.同源染色体2.同一染色体3.非同源染色体4.不同对染色体7、关于核糖体的移位，叙述正确的是（）1.空载tRNA的脱落发生在“A”位上2.核糖体沿mRNA的3’→5’方向相对移动3.核糖体沿mRNA的5’→3’方向相对移动4.核糖体在mRNA上一次移动的距离相当于二个核苷酸的长度8、Western blot是（）1.检测DNA的方法2.检测RNA的方法3.检测蛋白的方法4.检测酶的方法9、针对耐药菌日益增多的情况,利用噬菌体作为一种新的抗菌治疗手段的研究备受关注。

下列有关噬菌体的叙述,正确的是（）1.利用宿主菌的氨基酸合成子代噬菌体的蛋白质2.以宿主菌DNA为模板合成子代噬菌体的核酸3.外壳抑制了宿主菌的蛋白质合成,使该细菌死亡4.能在宿主菌内以二分裂方式增殖,使该细菌裂解10、a和b是不同顺反子的突变，基因型ab/++和a+/+b的表型分别为（）1.野生型和野生型2.野生型和突变型3.突变型和野生型4.突变型和突变型11、法医DNA科学涉及的学科有（）1.分子遗传学2.生物化学3.生物统计学4.以上都是12、下列哪种碱基不属于DNA/RNA的碱基（）1.腺嘌呤2.鸟嘌呤3.次黄嘌呤4.胸腺嘧啶13、要使两对基因杂合体自交后代群体纯合率达到96%以上，至少应该连续自交（）1.4代2.5代3.6代4.7代14、不属于质粒被选为基因运载体的理由是（）1.能复制2.有多个限制酶切点3.具有标记基因4.它是环状DNA15、关于朊蛋白（PrP）的叙述，下列哪项是错误的（）1.由人和动物细胞中的PrP基因编码2.由PrPC和PrPSC两种异构体3.PrPC有致病性和传染性4.PrPC对蛋白酶K敏感16、原核生物翻译过程中的起始氨基酸-tRNA是下列哪一项（）1.Met-tRNA2.fMet-tRNA3.Arg-tRNA4.Leu-tRNA17、在肺炎双球菌的转化实验中,使R型活菌转化成S型活菌的转化因子是（）1.荚膜2.蛋白质3.多糖4.S型细菌的DNA18、Northern印迹杂交是（）1.检测DNA的方法2.检测RNA的方法3.检测蛋白的方法4.检测酶的方法19、朊病毒样品经下列哪一种处理后，肯定失去了感染能力？ ( )1.100 ℃以上热处理2.电离辐射处理3.胰蛋白酶共温育15min4.进行氨基酸序列检测20、下列对DNA甲基化描述错误的是（）1.普遍存在原核生物和真核生物中2.能够调控基因的表达3.DNA甲基化改变了基因的序列4.由DNA甲基转移酶催化21、在乳糖操纵子中，与阻遏蛋白结合促进结构基因表达的是（）1.cAMP2.乳糖3.别构乳糖4.CAP22、Southern印记杂交是（）1.检测DNA的方法2.检测RNA的方法3.检测蛋白的方法4.检测酶的方法23、基因工程技术也称为DNA重组技术，其实施必须具备的四个必要条件是（）1.目的基因限制性内切酶运载体体细胞2.重组DNA; RNA聚合酶限制性内切酶连接酶3.工具酶目的基因运载体受体细胞4.模板DNA，信使RNA，质粒，受体细胞24、限制性内切酶的切割方式有（）1.5’粘性末端、3’粘性末端、平切2.5’粘性末端3.3’粘性末端4.平切25、用实验证实DNA半保留复制的学者是（）1.Watson和Crick2.Kornberg3.Nierenberg4.Meselson和Stahl26、1928年，格里菲斯通过肺炎双球菌实验首次证明了遗传物质是（）1.蛋白质2.DNA3.碳水化合物4.脂质27、在基因工程中通常所使用的质粒存在于（）1.细菌染色体2.酵母染色体3.细菌染色体外4.酵母染色体外28、将分离后的S型有荚膜肺炎双球菌的蛋白质外壳与R型无荚膜的肺炎双球菌混合注入小白鼠体内,小白鼠不死亡,从体内分离出来的依然是R型肺炎双球菌;将分离后的S型肺炎双球菌的DNA与R型肺炎双球菌混合注入小白鼠体内,则小白鼠死亡,并从体内分离出了S型有夹膜肺炎双球菌，以上实验说明了（）1.R型和S型肺炎双球菌可以相互转化2.R型的蛋白质外壳可诱导S型转化为R型3.S型的DNA可诱导R型转化为S型,说明了DNA是遗传物质4.R型的DNA可使小鼠死亡29、染色质DNA的碱基可被甲基化，DNA甲基化的作用是（）1.可关闭某些基因，同时可以活化另一些基因2.活化某些基因3.关闭某些基因4.与基因表达的调节无关30、下列哪一项不是真核生物mRNA的特点（）1.一般以单顺反子的形式存在2.前体合成后需经转录后加工3.半寿期短4.前体合成后无需转录后加工31、tRNA的二级结构为（）1.L状2.发卡结构3.三叶草形4.螺旋形32、基因工程的设计施工是在什么水平上进行的（）1.细胞2.细胞器3.分子4.原子33、真核生物RNA聚合酶中对α-鹅膏蕈碱十分敏感的是（）1.RNA聚合酶I2.RNA聚合酶II3.RNA聚合酶III4.RNA聚合酶IV34、DNA是主要的遗传物质是指（）1.遗传物质的主要载体是染色体2.大多数生物的遗传物质是DNA3.细胞里的DNA大部分在染色体上4.染色体在遗传上起主要作用35、下列哪项是正确的基因转录过程（）①核心酶识别终止子，释放RNA链；②DNA局部解链，第一个核苷酸结合到全酶上；③RNA聚合酶全酶与启动子结合；④核心酶在DNA链上移动；1.③②④①2.②④③①3.④①②③4.④②③①36、a和b是不同顺反子的突变，基因型ab/++和a+/+b的表型分别为（）1.野生型和野生型2.野生型和突变型3.突变型和野生型4.突变型和突变型37、真核生物转录所在的空间是（）1.细胞质2.细胞核3.核孔4.线粒体38、为什么使用限制性内切核酸酶对基因组DNA 进行部分酶切？（）1.为了产生平末端2.为了产生黏末端3.只切割一条链上的酶切位点，产生开环分子4.可得到比完全酶切的片段略长的产物39、下面关于限制性核酸内切酶的表示方法中，正确的一项是（）1.Sau3A2. E.coR3.hind III4.Sau3A I40、某科学工作者把兔子的血红蛋白的信使RNA加入到大肠杆菌的提取液中，在这个细胞的合成系统中能合成兔子的血红蛋白，这个事实说明（）1.蛋白质合成是在核糖体上进行的2.各种生物共用一套遗传密码3.兔子和大肠杆菌的基因发生了重组4.兔子的基因发生了突变41、细胞内合成DNA连接酶的场所是（）1.细胞核2.核区3.核糖体4.内质网42、天然的玫瑰没有蓝色花，这是由于缺少控制蓝色色素合成的基因B，而开蓝色花的矮牵牛中存在序列已知的基因B。

Chapter19Detection and Quantitative Analysis of Small RNAs by PCR Seungil Ro and Wei YanAbstractIncreasing lines of evidence indicate that small non-coding RNAs including miRNAs,piRNAs,rasiRNAs, 21U endo-siRNAs,and snoRNAs are involved in many critical biological processes.Functional studies of these small RNAs require a simple,sensitive,and reliable method for detecting and quantifying levels of small RNAs.Here,we describe such a method that has been widely used for the validation of cloned small RNAs and also for quantitative analyses of small RNAs in both tissues and cells.Key words:Small RNAs,miRNAs,piRNAs,expression,PCR.1.IntroductionThe past several years have witnessed the surprising discovery ofnumerous non-coding small RNAs species encoded by genomesof virtually all species(1–6),which include microRNAs(miR-NAs)(7–10),piwi-interacting RNAs(piRNAs)(11–14),repeat-associated siRNAs(rasiRNAs)(15–18),21U endo-siRNAs(19),and small nucleolar RNAs(snoRNAs)(20).These small RNAsare involved in all aspects of cellular functions through direct orindirect interactions with genomic DNAs,RNAs,and proteins.Functional studies on these small RNAs are just beginning,andsome preliminaryfindings have suggested that they are involvedin regulating genome stability,epigenetic marking,transcription,translation,and protein functions(5,21–23).An easy and sensi-tive method to detect and quantify levels of these small RNAs inorgans or cells during developmental courses,or under different M.Sioud(ed.),RNA Therapeutics,Methods in Molecular Biology629,DOI10.1007/978-1-60761-657-3_19,©Springer Science+Business Media,LLC2010295296Ro and Yanphysiological and pathophysiological conditions,is essential forfunctional studies.Quantitative analyses of small RNAs appear tobe challenging because of their small sizes[∼20nucleotides(nt)for miRNAs,∼30nt for piRNAs,and60–200nt for snoRNAs].Northern blot analysis has been the standard method for detec-tion and quantitative analyses of RNAs.But it requires a relativelylarge amount of starting material(10–20μg of total RNA or>5μg of small RNA fraction).It is also a labor-intensive pro-cedure involving the use of polyacrylamide gel electrophoresis,electrotransfer,radioisotope-labeled probes,and autoradiogra-phy.We have developed a simple and reliable PCR-based methodfor detection and quantification of all types of small non-codingRNAs.In this method,small RNA fractions are isolated and polyAtails are added to the3 ends by polyadenylation(Fig.19.1).Small RNA cDNAs(srcDNAs)are then generated by reverseFig.19.1.Overview of small RNA complementary DNA(srcDNA)library construction forPCR or qPCR analysis.Small RNAs are polyadenylated using a polyA polymerase.ThepolyA-tailed RNAs are reverse-transcribed using a primer miRTQ containing oligo dTsflanked by an adaptor sequence.RNAs are removed by RNase H from the srcDNA.ThesrcDNA is ready for PCR or qPCR to be carried out using a small RNA-specific primer(srSP)and a universal reverse primer,RTQ-UNIr.Quantitative Analysis of Small RNAs297transcription using a primer consisting of adaptor sequences atthe5 end and polyT at the3 end(miRTQ).Using the srcD-NAs,non-quantitative or quantitative PCR can then be per-formed using a small RNA-specific primer and the RTQ-UNIrprimer.This method has been utilized by investigators in numer-ous studies(18,24–38).Two recent technologies,454sequenc-ing and microarray(39,40)for high-throughput analyses of miR-NAs and other small RNAs,also need an independent method forvalidation.454sequencing,the next-generation sequencing tech-nology,allows virtually exhaustive sequencing of all small RNAspecies within a small RNA library.However,each of the clonednovel small RNAs needs to be validated by examining its expres-sion in organs or in cells.Microarray assays of miRNAs have beenavailable but only known or bioinformatically predicted miR-NAs are covered.Similar to mRNA microarray analyses,the up-or down-regulation of miRNA levels under different conditionsneeds to be further validated using conventional Northern blotanalyses or PCR-based methods like the one that we are describ-ing here.2.Materials2.1.Isolation of Small RNAs, Polyadenylation,and Purification 1.mirVana miRNA Isolation Kit(Ambion).2.Phosphate-buffered saline(PBS)buffer.3.Poly(A)polymerase.4.mirVana Probe and Marker Kit(Ambion).2.2.Reverse Transcription,PCR, and Quantitative PCR 1.Superscript III First-Strand Synthesis System for RT-PCR(Invitrogen).2.miRTQ primers(Table19.1).3.AmpliTaq Gold PCR Master Mix for PCR.4.SYBR Green PCR Master Mix for qPCR.5.A miRNA-specific primer(e.g.,let-7a)and RTQ-UNIr(Table19.1).6.Agarose and100bp DNA ladder.3.Methods3.1.Isolation of Small RNAs 1.Harvest tissue(≤250mg)or cells in a1.7-mL tube with500μL of cold PBS.T a b l e 19.1O l i g o n u c l e o t i d e s u s e dN a m eS e q u e n c e (5 –3 )N o t eU s a g em i R T QC G A A T T C T A G A G C T C G A G G C A G G C G A C A T G G C T G G C T A G T T A A G C T T G G T A C C G A G C T A G T C C T T T T T T T T T T T T T T T T T T T T T T T T T V N ∗R N a s e f r e e ,H P L CR e v e r s e t r a n s c r i p t i o nR T Q -U N I r C G A A T T C T A G A G C T C G A G G C A G GR e g u l a r d e s a l t i n gP C R /q P C Rl e t -7a T G A G G T A G T A G G T T G T A T A G R e g u l a r d e s a l t i n gP C R /q P C R∗V =A ,C ,o r G ;N =A ,C ,G ,o r TQuantitative Analysis of Small RNAs299 2.Centrifuge at∼5,000rpm for2min at room temperature(RT).3.Remove PBS as much as possible.For cells,remove PBScarefully without breaking the pellet,leave∼100μL of PBS,and resuspend cells by tapping gently.4.Add300–600μL of lysis/binding buffer(10volumes pertissue mass)on ice.When you start with frozen tissue or cells,immediately add lysis/binding buffer(10volumes per tissue mass)on ice.5.Cut tissue into small pieces using scissors and grind it usinga homogenizer.For cells,skip this step.6.Vortex for40s to mix.7.Add one-tenth volume of miRNA homogenate additive onice and mix well by vortexing.8.Leave the mixture on ice for10min.For tissue,mix it every2min.9.Add an equal volume(330–660μL)of acid-phenol:chloroform.Be sure to withdraw from the bottom phase(the upper phase is an aqueous buffer).10.Mix thoroughly by inverting the tubes several times.11.Centrifuge at10,000rpm for5min at RT.12.Recover the aqueous phase carefully without disrupting thelower phase and transfer it to a fresh tube.13.Measure the volume using a scale(1g=∼1mL)andnote it.14.Add one-third volume of100%ethanol at RT to the recov-ered aqueous phase.15.Mix thoroughly by inverting the tubes several times.16.Transfer up to700μL of the mixture into afilter cartridgewithin a collection bel thefilter as total RNA.When you have>700μL of the mixture,apply it in suc-cessive application to the samefilter.17.Centrifuge at10,000rpm for15s at RT.18.Collect thefiltrate(theflow-through).Save the cartridgefor total RNA isolation(go to Step24).19.Add two-third volume of100%ethanol at RT to theflow-through.20.Mix thoroughly by inverting the tubes several times.21.Transfer up to700μL of the mixture into a newfilterbel thefilter as small RNA.When you have >700μL of thefiltrate mixture,apply it in successive appli-cation to the samefilter.300Ro and Yan22.Centrifuge at10,000rpm for15s at RT.23.Discard theflow-through and repeat until all of thefiltratemixture is passed through thefilter.Reuse the collectiontube for the following washing steps.24.Apply700μL of miRNA wash solution1(working solu-tion mixed with ethanol)to thefilter.25.Centrifuge at10,000rpm for15s at RT.26.Discard theflow-through.27.Apply500μL of miRNA wash solution2/3(working solu-tion mixed with ethanol)to thefilter.28.Centrifuge at10,000rpm for15s at RT.29.Discard theflow-through and repeat Step27.30.Centrifuge at12,000rpm for1min at RT.31.Transfer thefilter cartridge to a new collection tube.32.Apply100μL of pre-heated(95◦C)elution solution orRNase-free water to the center of thefilter and close thecap.Aliquot a desired amount of elution solution intoa1.7-mL tube and heat it on a heat block at95◦C for∼15min.Open the cap carefully because it might splashdue to pressure buildup.33.Leave thefilter tube alone for1min at RT.34.Centrifuge at12,000rpm for1min at RT.35.Measure total RNA and small RNA concentrations usingNanoDrop or another spectrophotometer.36.Store it at–80◦C until used.3.2.Polyadenylation1.Set up a reaction mixture with a total volume of50μL in a0.5-mL tube containing0.1–2μg of small RNAs,10μL of5×E-PAP buffer,5μL of25mM MnCl2,5μL of10mMATP,1μL(2U)of Escherichia coli poly(A)polymerase I,and RNase-free water(up to50μL).When you have a lowconcentration of small RNAs,increase the total volume;5×E-PAP buffer,25mM MnCl2,and10mM ATP should beincreased accordingly.2.Mix well and spin the tube briefly.3.Incubate for1h at37◦C.3.3.Purification 1.Add an equal volume(50μL)of acid-phenol:chloroformto the polyadenylation reaction mixture.When you have>50μL of the mixture,increase acid-phenol:chloroformaccordingly.2.Mix thoroughly by tapping the tube.Quantitative Analysis of Small RNAs3013.Centrifuge at10,000rpm for5min at RT.4.Recover the aqueous phase carefully without disrupting thelower phase and transfer it to a fresh tube.5.Add12volumes(600μL)of binding/washing buffer tothe aqueous phase.When you have>50μL of the aqueous phase,increase binding/washing buffer accordingly.6.Transfer up to460μL of the mixture into a purificationcartridge within a collection tube.7.Centrifuge at10,000rpm for15s at RT.8.Discard thefiltrate(theflow-through)and repeat until allof the mixture is passed through the cartridge.Reuse the collection tube.9.Apply300μL of binding/washing buffer to the cartridge.10.Centrifuge at12,000rpm for1min at RT.11.Transfer the cartridge to a new collection tube.12.Apply25μL of pre-heated(95◦C)elution solution to thecenter of thefilter and close the cap.Aliquot a desired amount of elution solution into a1.7-mL tube and heat it on a heat block at95◦C for∼15min.Open the cap care-fully because it might be splash due to pressure buildup.13.Let thefilter tube stand for1min at RT.14.Centrifuge at12,000rpm for1min at RT.15.Repeat Steps12–14with a second aliquot of25μL ofpre-heated(95◦C)elution solution.16.Measure polyadenylated(tailed)RNA concentration usingNanoDrop or another spectrophotometer.17.Store it at–80◦C until used.After polyadenylation,RNAconcentration should increase up to5–10times of the start-ing concentration.3.4.Reverse Transcription 1.Mix2μg of tailed RNAs,1μL(1μg)of miRTQ,andRNase-free water(up to21μL)in a PCR tube.2.Incubate for10min at65◦C and for5min at4◦C.3.Add1μL of10mM dNTP mix,1μL of RNaseOUT,4μLof10×RT buffer,4μL of0.1M DTT,8μL of25mM MgCl2,and1μL of SuperScript III reverse transcriptase to the mixture.When you have a low concentration of lig-ated RNAs,increase the total volume;10×RT buffer,0.1M DTT,and25mM MgCl2should be increased accordingly.4.Mix well and spin the tube briefly.5.Incubate for60min at50◦C and for5min at85◦C toinactivate the reaction.302Ro and Yan6.Add1μL of RNase H to the mixture.7.Incubate for20min at37◦C.8.Add60μL of nuclease-free water.3.5.PCR and qPCR 1.Set up a reaction mixture with a total volume of25μL ina PCR tube containing1μL of small RNA cDNAs(srcD-NAs),1μL(5pmol of a miRNA-specific primer(srSP),1μL(5pmol)of RTQ-UNIr,12.5μL of AmpliTaq GoldPCR Master Mix,and9.5μL of nuclease-free water.ForqPCR,use SYBR Green PCR Master Mix instead of Ampli-Taq Gold PCR Master Mix.2.Mix well and spin the tube briefly.3.Start PCR or qPCR with the conditions:95◦C for10minand then40cycles at95◦C for15s,at48◦C for30s and at60◦C for1min.4.Adjust annealing Tm according to the Tm of your primer5.Run2μL of the PCR or qPCR products along with a100bpDNA ladder on a2%agarose gel.∼PCR products should be∼120–200bp depending on the small RNA species(e.g.,∼120–130bp for miRNAs and piRNAs).4.Notes1.This PCR method can be used for quantitative PCR(qPCR)or semi-quantitative PCR(semi-qPCR)on small RNAs suchas miRNAs,piRNAs,snoRNAs,small interfering RNAs(siRNAs),transfer RNAs(tRNAs),and ribosomal RNAs(rRNAs)(18,24–38).2.Design miRNA-specific primers to contain only the“coresequence”since our cloning method uses two degeneratenucleotides(VN)at the3 end to make small RNA cDNAs(srcDNAs)(see let-7a,Table19.1).3.For qPCR analysis,two miRNAs and a piRNA were quan-titated using the SYBR Green PCR Master Mix(41).Cyclethreshold(Ct)is the cycle number at which thefluorescencesignal reaches the threshold level above the background.ACt value for each miRNA tested was automatically calculatedby setting the threshold level to be0.1–0.3with auto base-line.All Ct values depend on the abundance of target miR-NAs.For example,average Ct values for let-7isoforms rangefrom17to20when25ng of each srcDNA sample from themultiple tissues was used(see(41).Quantitative Analysis of Small RNAs3034.This method amplifies over a broad dynamic range up to10orders of magnitude and has excellent sensitivity capable ofdetecting as little as0.001ng of the srcDNA in qPCR assays.5.For qPCR,each small RNA-specific primer should be testedalong with a known control primer(e.g.,let-7a)for PCRefficiency.Good efficiencies range from90%to110%calcu-lated from slopes between–3.1and–3.6.6.On an agarose gel,mature miRNAs and precursor miRNAs(pre-miRNAs)can be differentiated by their size.PCR prod-ucts containing miRNAs will be∼120bp long in size whileproducts containing pre-miRNAs will be∼170bp long.However,our PCR method preferentially amplifies maturemiRNAs(see Results and Discussion in(41)).We testedour PCR method to quantify over100miRNAs,but neverdetected pre-miRNAs(18,29–31,38). 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2025高考英语一轮复习外刊阅读与词汇专练专题04 RNA破防了！我不是DNA的小弟！1. 精编外刊阅读2. 阅读理解专项3. 语法填空专项4. 课标高频词专练5. 外刊中的课标词【精编·外刊阅读】A primer on RNA, perhaps the most consequential molecule of all（文章来源：Economist）文中红色粗体为课标词，下面有专门的高频课标词训练和课标词梳理表格For years, students of cellbiology were taught that RNA wasmerely a humble assistant to DNAand proteins. DNA was seen as thelibrary of all knowledge and proteinsas the constructors of an organism.RNA was viewed as a messenger（信使）, carrying DNA's plans tocell workshops and being part of theworkshop fabric. Biologists now realize that RNA has a far wider range of jobs in cells than earlier understood. It seems likely that RNA even precedes DNA and proteins as the original molecule（分子）of life.Thomas Cech's new book, "The Catalyst," describes how the view of RNA has changed. In the 1980s, Cech supported the idea that RNA molecules can act as enzymes（酶）, challenging the belief that only proteins could be catalysts. In 1989, he shared the Nobel chemistry prize for discovering "ribozymes （核酶）". Dr Cech’s team found an "autocatalytic（自催化的）" rearrangement of an RNA molecule. This molecule, meant to bee part of a ribosome（核糖体）, cut out an unnecessary part. This discovery challenged the belief that enzymes are always proteins.Similar discoveries by other labs quickly followed, revealing other types of ribozymes. RNA in ribosomes was discovered to be catalytic, not just structural. It is RNA, not the protein ponent, that adds amino（氨基） acids to a growing protein chain. This discovery excited scientists seeking life’s origin. RNA, which can both store information and catalyze（催化）reactions, may have been the earliest molecule of life. Early RNAbased organisms may have later evolved to use DNA for information storage and proteins for catalysis, with RNA linking these molecules.Since Dr Cech’s discovery, many types of RNA have been found, involved in gene regulation and protecting cells from viral infection. About half of medicines work by targeting germ RNA while leaving human RNA unaffected, which is a promising starting point for new drugs. RNA can silence disease causing genetic changes by pairing with and disabling RNA messengers from changed DNA sections. RNA messengers have been used to create covid vaccines and may be used against other diseases, including certain cancers.【原创阅读理解】1.What was RNA traditionally viewed as in cell biology?A. A primary molecule responsible for genetic inheritanceB. A secondary molecule assisting DNA and proteinsC. The main structural ponent of cells and tissuesD. An enzyme that catalyzes biochemical reactions2.How can the word "catalysts" be interpreted in the context of this passage?A. Things that slow down chemical reactions in cellsB. Proteins that support and maintain cell structuresC. Molecules that carry genetic information to cellsD. Substances that help speed up chemical reactions3.Why is RNA important in the study of life's origin?A. RNA's ability to act as both genetic material and an enzyme supports theories of early lifeB. RNA's stability and versatility make it essential for understanding early lifeC. RNA's simplicity pared to DNA and proteins suggests it was the first biological moleculeD. RNA's presence in early organisms underscores its evolutionary importance4.What does the article imply about the future possibilities for RNA in medicine?A. RNA will likely bee the main focus of genetic research, overshadowing DNAB. RNAbased therapies have the potential to revolutionize treatment for various diseasesC. RNA's role in cellular functions suggests it will replace proteins in many therapiesD. RNA applications are limited, but they show promise in specialized fields like oncology【原创语法填空】For years, students of cell biology were taught that RNA was merely an assistant to DNA and proteins. DNA ____1____ (consider) the library of all knowledge, and proteins were seen as the buildersof an organism. RNA was viewed as a messenger, ____2____ (carry) DNA's instructions to cell workshops. Biologists now realize that RNA performs a much ____3____ (wide) range of jobs in cells.Thomas Cech's book, "The Catalyst," highlights how perceptions of RNA have changed. In the 1980s, Cech proposed that RNA molecules can act as enzymes, challenging the belief that only proteins could be catalysts. In 1989, he won the Nobel Prize for discovering "ribozymes." His team identified____4____ "autocatalytic" RNA molecule ____5____ removed an unnecessary part to bee part of a ribosome.Other labs quickly made similar ____6____ (discovery), identifying more ribozymes. RNA in ribosomes was found to be catalytic, not just structural. It is RNA, not protein, ____7____ adds amino acids to a growing protein chain. RNA, capable of storing information and catalyzing reactions, may have been the earliest molecule of life. Early RNAbased organisms might have evolved to use DNA for information storage and proteins for catalysis, ____8____ RNA linking these molecules.About half of medicines work by targeting germ RNA while leaving human RNA unaffected. RNA messengers ____9____ (use) to create COVID19 vaccines and might be used against other diseases,____10____ (include) cancers.【原创·课标高频词训练】1.It is __________ (necessary) to provide further proof when the evidence is already overwhelming.2.Our current __________ (store) capabilities are insufficient for the volume of data we handle daily.3.The government's new __________ (regulate) on emissions has sparked controversy among carmanufacturers.4.Over millions of years, animals __________ (evolve) specialized traits to survive in their habitats.5.The campaign __________ (target) demographic includes young adults aged 1825.6.Scientists constantly __________ (seek) to understand the underlying causes of plex diseases.7.The study __________ (reveal) significant differences between the two groups.8.The temperature __________ (range) in this region can vary dramatically between day and night.9.The __________ (origin) manuscript of the novel is preserved in the national library.10.The mittee is __________ (mere) advisory and has no decisionmaking powers.11.Given the current circumstances, it is highly __________ (like) that the project will be delayed.12.The investigation __________ (involve) multiple agencies working collaboratively.13.Proper hygiene practices can significantly reduce the risk of __________ (infect).14.The project presents many __________ (challenge) to the team, requiring innovative solutions.15.The patient's __________ (react) to the medication was carefully monitored by the doctors.【梳理·外刊中的课标词】。

一种莱茵衣藻耐铬突变体及其应用一种莱茵衣藻耐铬突变体及其应用莱茵衣藻（Chlamydomonas reinhardtii）是一种单细胞的绿色海藻，广泛存在于清澈的淡水湖泊和河流中。

它们是理想的实验材料，因为它们生活史快，繁殖能力强，易于培养和遗传改造。

最近，一种莱茵衣藻的耐铬突变体被发现并引起了科学家们的极大关注。

第一步：发现莱茵衣藻耐铬突变体研究人员使用高浓度的六价铬（Cr (VI)）对莱茵衣藻进行了处理，研究表明，莱茵衣藻能够从培养基中释放三价铬（Cr (III)），这是一种比较安全的代谢方式。

然而，高剂量的六价铬将会对细胞产生毒害，导致严重的生长抑制和细胞死亡。

研究人员培养了一组莱茵衣藻并将其暴露于高浓度的六价铬中，随着时间的推移，细胞很快死亡了，但是其中有一些细胞并未被毒害。

通过对这些细胞的基因组测序，科学家们发现了一个潜在的突变位点，它在一些细胞上被发现了，而在其他细胞上则未出现。

经过验证，他们发现，这种突变导致莱茵衣藻在高浓度的六价铬环境下仍然能够生存，成活率达到了极高的水平。

这样的发现被认为是莱茵衣藻属于铬-还原细菌的证据之一，在不同的环境条件下，莱茵衣藻在代谢铬时有不同的适应性。

第二步：应用这种铬耐受性，可以在环境净化技术和饮用水净化技术中得到应用。

经过遗传改造的莱茵衣藻可以被运用在化工和冶金领域，如酸性矿山废水处理、污染土壤修复等领域，有望为解决环境污染问题提供新的技术支持。

此外，这项技术还可以用于生产莱茵衣藻作为饲料、食品和生物燃料原料。

此外，该技术还可以用于开发针对医学领域的互补性治疗。

这意味着这项技术不仅可以解决环境问题，而且还具备广阔的商业应用前景。

总结：综上所述，这种莱茵衣藻的耐铬突变体及其应用在环境净化技术和生物技术领域具有广泛的应用前景和深远的意义，它不仅为清理环境和绿色发展提供了便利，而且可以促进生物科技的进一步发展，具备巨大的社会和经济效益。

未来，我们还可以将这种技术应用到更广泛的领域，来推动人类社会的可持续发展。

乙酸钠为基质的微生物燃料电池产电过程研究冯玉杰*，王鑫，李贺，（哈尔滨工业大学城市水资源与水环境国家重点实验室，哈尔滨，150090）摘要:以多孔碳纸为阳极，耐水性电催化材料为阴极，设计了无媒介双室微生物燃料电池（Microbial Fuel Cell,MFC）。

以厌氧污泥为出发菌株，乙酸钠为底物，外接一定负载条件下，进行了MFC产电过程研究。

分别研究了进水浓度在800，1200，1600，2000mg/L，以及在外电阻条件为400、600、800、1000Ω，水力停留时间48h时，负载两端的电压、功率密度、以及电化学池中生物量（VSS）和出水COD的变化规律。

结果表明，进水浓度升高时，阳极池内生物量减少，COD去除率降低，MFC功率密度提高。

在进水乙酸钠浓度2000mg/L时，MFC最高功率密度35.71mW/m2，电流密度345mA/m2。

外电阻阻值降低后，平均出水COD升高，MFC电流升高，阳极池微生物产电能力增强。

关键词:微生物燃料电池;生物产电；废水处理微生物燃料电池（Microbial Fuel Cell,MFC）是一种利用微生物的催化作用，将燃料中的化学能转化为电能的电化学装置，如图1所示[1]，该装置以阳极溶液中蔗糖、醋酸盐等有机物[2,3]作为燃料，在微生物的作用下从燃料中获得电子并传递到阳极，电子通过外电路到达阴极。

同时将产生的质子通过质子交换膜传递到阴极。

在阴极，电子、氧化剂和质子反应生成还原产物，完成整个生物电化学过程和能量转化过程。

英国植物学家Potter于1911年用酵母和大肠杆菌进行试验，首次发现利用微生物可以产生电流[4]，但由于氧化还原媒介体（中性红、亚甲基蓝、劳氏紫等）价格昂贵并且一部分具有毒性，阻碍了微生物燃料电池的发展。

二十世纪初人们逐渐发现一些微生物可以不通过氧化还原媒介体直接氧化有机物转移电子，并以Fe（Ⅲ）为最终电子受体[5,8]。

随后的研究表明，诸如Geobacter sulfurreducens，Shewanella putrefacien IR-1和Clostridium butyricum EG3等微生物[6-8]均具有电化学活性，它们能够在没有外加氧图1微生物燃料电池结构示意图化还原媒介体的条件下将电子从底物转移到阳极板上，自发的产生电流。

多电荷DNA框架介导的外泌体microRNA高灵敏电化学检测多电荷DNA框架介导的外泌体microRNA高灵敏电化学检测随着科技的不断进步，外泌体(microvesicles)作为传递细胞信息的重要方式，受到了广泛的研究关注。

外泌体中携带的microRNA，作为一类重要的非编码RNA分子，参与了许多生物学过程，如细胞信号传导、基因调控等。

因此，研究外泌体中的microRNA对于探究疾病的发生机制、诊断和治疗具有重要意义。

然而，由于外泌体中microRNA含量稀少且高度异质性，传统的检测方法往往难以满足高灵敏度、高选择性和高通量等要求。

因此，如何实现对外泌体中microRNA的高灵敏检测成为了当前研究的热点之一。

近年来，多电荷DNA框架在电化学传感领域展现出了广阔的应用前景。

多电荷DNA框架是一类具有多个正电荷的DNA序列，其特殊的序列结构和电荷特性使其在电化学检测中具有良好的信号放大效应和高灵敏度。

基于多电荷DNA框架的电化学传感技术已经在生物医学领域中取得了一系列的突破。

在外泌体中microRNA的检测中，多电荷DNA框架可以作为一个理想的信号放大器。

当外泌体中的microRNA与多电荷DNA框架结合时，多电荷DNA框架的电荷特性可以增强microRNA与电极表面的相互作用，从而提高电化学信号的灵敏度。

为了实现高灵敏的电化学检测，研究人员通过合理设计多电荷DNA框架的序列结构，以增强与外泌体中microRNA的结合亲和力。

同时，结合纳米材料技术，如金纳米颗粒、石墨烯等，可以在电极表面导入更多的多电荷DNA框架，进一步提高电化学信号的放大效应。

此外，通过优化电化学检测方法的条件，如pH值、电位等，也可以提高电化学检测的灵敏度。

除了高灵敏度外，高选择性也是外泌体中microRNA电化学检测的重要指标。

多电荷DNA框架的序列特异性可以通过合理设计和改变多电荷DNA框架的序列来实现。

例如，通过引入特定的杂交位点或化学修饰物，可以实现对目标microRNA的选择性识别。

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