生物工程-Concepter-of-Gene教学讲解

1、The age of complete genomic sequencesThe more we learn about genes, the less sure we are of what a gene really is WHAT ARE GENES CHAPTER TWOThe Concept of the Gene in Development and Evolution The hereditary nature of every living organism is defined by its genome, which consists of a long sequence

2、of nucleic acid that provides the information needed to construct the organism.每个生物体的基因组决定了该生物体的遗传特性，基因组拥有很长的核苷酸序列，能提供组建机体所需的信息。A few facts about inheritance known since ancient times: Children resemble their parents. Domestication followed by selective breeding to improve plants and animals. Betwee

3、n 8000 and 1000 B.C.赫伯特斯宾塞（Herbert Spencer , 1820-1903）： physiological units 查里斯达尔文（ Charles Darwin，1809-1882 ）： gemmules奥古斯特魏斯曼（ August Weismann,1834 - 1914)： germplasm1. Early gene concept2. Classical gene concept3. Gene concept evolution and development4. DNA is the main genetic material5. Molecu

4、lar configuration of gene6. Gene concept multiplicity1. Early gene concept1.1 The Greek Influence on ideas of inheritanceHippocrates “Humors”, which could be altered during an individuals lifetime and therefore diseased or normal, were drawn from various parts of the body to the semen and passed on

5、to the offspring. This “pangenesis” theory even formed the basis of Darwins early ideas of inheritance.Aristotle semen produced a “vital heat” that cooked and shaped the menstrual blood giving it the capacity to produce offspring with the same “form” as the parent.Later ideas of inheritance (1600- 1

6、850)Preformationism is the idea that each sperm contains a miniature person : development is merely a process of enlarging and maturing the person already present in the sperm cell. 11Epigenesis presumably put forth by Harvey, held that body structures were not initially present in the sex cells, bu

7、t instead formed de novo (anew) .Illustration of homunculi in sperm, drawn by Hartsoeker in 1695Later ideas of inheritance (1600- 1850)11Blending Inheritance the belief that characteristics of parents blended like mixing red paint with white paint: the results is pink paint, and there is no way to e

8、ver separate out the red and white. Example of blending inheritance using the color of flowers to show how a species color variation would converge upon one color in relatively few generations if its offsprings color variations were truly bounded by the parents colors. 1.2 获得性遗传理论（Inheritance of acq

9、uired characters L.B.Lamarck. 1809） Nature produced successively all the different forms of life on earth. Environmentally induced behavioral changes lead the way in species change.More recently, “epigenetic inheritance” has been represented as a form of the transmission of acquired characters and t

10、hus as a confirmation, at least of a sort, of Lamarcks most famous idea.1.3 泛生论假说 （Hypothesis of the Pangenesis） Variations in Animals and Plants under Domestication 1.4 种质论（Theory of germplasm A.Weismann 1883. ）GermplasmSomatoplasmRoot, Stem, LeafSomatoplasm Germplasm GermplasmExperiments in Plant

11、Hybridization 1.5 遗传因子假说 (Hypothesis of the inherited factor G. J. Mendel 1866. ) 生物性状由遗传因子控制 亲代传给子代的是遗传因子(A,a.) 遗传因子在体细胞内成双(AA，aa) 在生殖细胞内为单（A，a） 杂合子后代体细胞内具有成双的遗传因子(Aa) 等位的遗传因子独立分离 非等位遗传因子间自由组合地分配到配子中否定了 Hypothesis of the Pangenesis（泛生论 ） 奠定了 Theory of particulate inheritance 提出了 Law of segregation

12、Law of independent assortment 缺乏细胞在有丝分裂和减数分裂过程中 染色体行为的有力证据。 每一个遗传因子是一个相对独立的功能单位 遗传因子的等位性 遗传因子的纯洁性Gregor Mendel 1822-1884My time will come The work was simply too ahead of its time, too contrary to popular beliefs about heredity. My time will come, Mendel once said, but it was over 30 years before hi

13、s work was appreciated.1900Carl Correns（1864-1933）德国 图宾根大学peas Principle of independent segregationErich von Tschermark（1871-1962）奥地利 维也纳农业大学peasMendels principles were independently discovered and verified, marking the beginning of modern genetics. Hugo de vries（1848-1935）荷兰 阿姆斯特丹大学evening primrose

14、Principle of independent assortmentWilhelm Johannsen (1857 - 1927) 1909 Johannsen proposed the term gene to describe Mendels factors of inheritance.但究竟细胞中什么结构与遗传因子相一致?细胞学与遗传学的结合为解决这一问题提供了途径。The Chromosome Theory of Heredity 二倍体生物减数分裂后每个配子仅接受成对染色体中的一个。 假定基因是染色体的一部分。 第一次将遗传学和细胞学科学地结合起来 为Mendel遗传规律的解释提

15、供了细胞学证据Walter Sutton (18771916) Theodor Heinrich Boveri (1862 1915) Boveri was studying sea urchins(海胆), in which he found that all the chromosomes had to be present for proper embryonic development to take place. Suttons work with grasshoppers(蝗虫) showed that chromosomes occur in matched pairs of m

16、aternal and paternal chromosomes which separate during meiosis and may constitute the physical basis of the Mendelian law of heredity .由于同源染色体的分离而实现等位基因的分离，导致性状的分离；决定不同性状的两对非等位基因分别处在两对非同源染色体上，由于同源染色体的分离、非同源染色体的独立分配，导致了基因的自由组合。必须进一步把某一特定基因与特定染色体相联系，证明基因的行为与染色体在细胞分裂中行为的平行关系转变为基因与染色体的从属关系。 There are ex

17、ception to Mendels lawsIncomplete dominance.Codominance.Linked Genes.Linked Genes On The Same Chromosome Exhibit Distorted Mendelian Ratios - In 1905, William Bateson, Edith Rebecca Saunders, and Reginald Punnett P genenration Homozygous strainsPurple flowers long pollenRed flowers round pollenF1 ge

18、nenration Purple flowers long pollenF2 genenration1528Purple flowers long pollen106Purple flowers roud pollen118Red flowers long pollen381Red flowers round pollenPhenotypeExpectedObservedPurple, long11991528Purple, round400106Red, long400118Red, round133381Total21322132F2 9：3：3：1 2. 经典的基因概念 Theory o

19、f the gene(1925) 基因是染色体上的实体 基因象链珠一样，孤立地呈线状地排列在染色体上 基因是 （Three in one） 功能(functional unit)突变(mutation unit) 交换(cross-over unit) “三位一体”的 最小的 不可分割的基本的遗传单位 Thomas Hunt Morgan(1866 1945)1866. -1926. 经典遗传学理论 3. 基因概念的演 变与发展 3.1 基因的位置效应 Position effectAlfred Henry Sturtevant (18911970 )基因的表达要求转录因子与调控顺序结合，这不

20、仅与核小体的状态有关，还涉及基因所在的染色质区段的高级结构。当染色质处在致密收缩状态时，转录因子无法与染色体包裹的DNA结合，基因被关闭。这种因染色体不同区段结构而影响基因表达的现象称为位置效应。稳定位置效应（stable position effect）花斑位置效应（variegated position effect） Bar eye Position effect Dosage effect Duplication Wild type16A779个68个45个chromosome：16区A段385个 稳定位置效应（stable position effect） 花斑位置效应（ Posit

21、ion-effect variegation，PEV） 常由易位而引起。这种位置效应与异染色质的影响有关。基因活性依赖于所处染色体位置。Elgin SC,Reuter G. Cold Spring Harb Perspect Biol.2013 Aug 1;5(8):a017780.A schematic illustration ofwhitevariegation in the X chromosome inversionIn(1)wm4.Slotkin RK, Martienssen R(2007).Transposable elements and the epigenetic reg

22、ulation of the genome. Nature Reviews Genetics 8, 272-285The influence of transposable elements on gene expressionPosition effect variegation (PEV) on Drosophila melanogaster chromosome 4 correlates with the proximity of a transgene to a transposable element (TE).Three examples of variegation induce

23、d by TE-generated epialleles.stwl mutants are dominant suppressors of position-effect variegation. A. Eye of DX1 flies showing strong variegation. B,C. Eyes of DX1 flies also heterozygous for stwl alleles showing suppressed variegation. D. The DX1 variegated phenotype was dominantly suppressed by a

24、mutation in the histone methyltransferase encoded by the Su(var)3-9 locus. E. Graph of optical density of extracted pigments from flies of the indicated genotypes. Maines JZ,Park JK,Williams M,McKearin DM(2007). Stonewalling Drosophila stem cell differentiation by epigenetic controls. Development 13

25、4, 1471-1479. 3.2 拟等位基因概念的提出 （pseudo alleles） Multiple alleles：在群体中，同一基因座上具有三个或三个以上的等位基因。 a1A a2 a3Aa1a2a1a1 a2a2 a1a2 ( no wild type ) But !W w- wa red eye (w.t) w-wa w-Y Xw- Y Xw- Xw- Xw- Xw- Y Xwa Xw- Xwa XwaY ?1/1000W.Tred eyewhite eye (mut) amygdaloid eye (mut) in DrosophilaX w YXwXw X wXw YXwX

26、w X wXw YXw YXwXw XwXw YX wXw X wXw YX w+w YXww+Xww+ Xw+ w Xww+ YXww+Xww+X w+w Xww+Y?1/1000W.Tred eye杏色眼基因和白眼基因在染色体上所占的位置相同，即位于同一基因座，但属于不同位点，因而它们之间可以发生交换。 Xw w+ YXw w+ Xw w+ Xw w+ Xw w+ YX w+w X w+w Xw w+ X w+w YX w+ w+ X w+ w+ Xw w+ X w+ w+ YXw w Xw w Xw w+ Xw w Yww+w+w杏色眼w+w+ww红色眼trans cis 这种由于排列方

27、式不同而表型不同的现象称为cis-trans position effects，并将这种紧密连锁的功能性等位基因，但不是结构性的等位基因称为pseudo allele。拟等位基因的发现也证明基因的可分性。 紧密连锁 （交换率极低），功能相同（表型相似） Genetics developing Pseudo Alleles From 1940s, Microbe as genetic research material A1 A2 a1 a2 w.tMut.A1 a2 a1 A2 SingleSimplePropagationPopulation 3.3 顺反子理论（Theory of cist

28、ron） (S.Benzer 1955) 1941 Beadle & TatumNuraspora crassa One gene -one enzyme 1944 Oswald Avery Diplococcum pneumonice DNA as genetic material 1960 Jacob & MonodEscherichia coli Lactose operon 1955 S.BenzerE. coli T4 phage Cistron 在这一特定的DNA片段内含有许多突变位点（muton），即突变后可产生变异的最小单位。突变位点之间可以发生重组，因此一个基因内含有多个重组

29、单位（recon），即不能由重组分开的最小单位。基因是DNA分子上一个特定的区段，就其功能来说是一个独立单位。打破了“三位一体”的基因概念，把基因具体为DNA分子上特定的一段顺序，即负责编码特定遗传信息的功能单位顺反子。包含多个突变和重组单位。噬菌体的突变型 噬菌斑形态的突变型 寄主范围的突变型条件致死突变型 Mut.T4 rII : rII107, rII105, rII51, rII47(3000) PhageE.coli BE.coli k12W.t T4Small turbid plaque Small turbid plaque Mut.T4 rIIBig clear plaque

30、都有相同的表型，那么这3000多个突变型是否都影响同一种遗传功能？也就是说它们是属于一个基因还是几个基因？ Recombination assay & Complementary assay rII47 0 0 rII102 E.coli B plane E.coli B Blotting into plane E.coli K12 （） Recombination assay （重组测验） 0 0 rII47 rII104 一份接种B菌株，在B菌株细胞中r47+、+r104、r47r104、+都能生长，因此在此平板上可统菌体的总数另一份接种于K()菌株中，只有+重组子长，由于r双重突变的交互

31、重组子r47r104不能生长，所以无法检出，但是它频率和+相等，因此估算重组子数要把+数乘以2，代入公式：重组频率=2 r+噬菌斑数噬菌斑数总数 100=2大肠杆菌K()菌株上的噬菌斑数 大肠杆菌B菌株上的噬菌斑数总数 100利用两个r不同突变型如 r47+和 + r104 在许可条件下进行双重感染，即同时侵染大肠杆菌B菌株，形成噬菌斑后收集溶菌液，将此溶菌液等分两份即使在106 r噬菌体中只出现一个r+重组子，也可通过感染大肠杆菌K()菌株的平板检查出来，因此根据公式在理论上可测到两个r突变之间重组频率为0.002(2x102/106=2x10-4)以遗传图距的方式确定突变子之间的空间关系。

32、实际所观察的最小重组频率为0.02，即0.02个图距单位，还没有发现小于这个数值的重组频率T4染色体有1.8105个核苷酸对，其长度为1500个图距单位，因此0.02个图距单位约等于2个核苷酸对1.0r47 r104 r101 r103 r105 r106 r51 r1021.32.20.60.51.01.51.92.81.61.41.11.41.82.05.02.23.92.72.95.8Three in one ! RII region RII 47 104 101 103 105 106 51 102 How many genes ?！ 0 0 rII47 rII106 0 0 rII1

33、06 rII51 Complementary assay plane E.coli K12()recombination ? ! function complementaryrII 47 rII106rII106 rII51difficult propagationrecombinationWhy?How? A b1 A b2 a1 BA b2相 依 为 命 ！依据：One gene one enzyme Mutant Wild type 无 能 为 力 ！杂合二倍体内，野生型基因对突变型等位基因,可以发生功能的补偿, 产生功能互补效应。带有不同突变位点的噬菌体同时感染一个E.coli，构成双

34、突变杂合二倍体，组成互补测验体系，以测定各突变位点所在基因的等位性。A b1 A b2 a1 BA b2不同的非等位基因 同一等位基因 功能互补效应的测验体系具有 不具有 突变位点 处于 a1 BA b2A b1 A b2 + -+ +ABABBAAB顺式测验 反式测验 突变发生在同一顺反子内 突变发生在不同顺反子内 Benzer的互补测验中所用的两个突变型，分别位于两条染色体上，这种组合方式称为反式排列，如果两个突变同时位于一条染色体上，则称为顺式排列。在互补测验中，两个隐性突变如表现出互补效应，则证明这两个突变型分别属于不同基因；如不能表现出互补，则证明这两个突变型是在同一基因内。 rII

35、47 104 101 103 105 106 51 102 A gene B gene rII of T4 phage including two genes Theory of cistron Cistron 是基因的同义词 在一个顺反子内，有若干个muton 在一个顺反子内，有若干个recon基因是具有特定功能、完整的、不可分割的最小遗传单位 基因内可以较低频率发生基因内的重组，交换 pseudo alleles 是基因内的突变体 mut1 X mut2 W.t 是基因内发生交换的结果 3.4 等位基因（Allele, Allomorph）概念的发展Pseudo alleleDNA多型性检

36、测技术的发展 ( RFLP, RAPD ) Allele: 同一座位存在的两个以上不同状态的基因, 其总和称之为复等位基因（multiple alleles）全同等位基因 (homoallele) 非全同等位基因 (heteroallele) Allele 具有相对差异的DNA区域 TAAAGTAAT TAAAGCAAT site Gene locus Mut a1 Mut a2W.t AMut a1Mut a2ATTCTGAGCTATTCGGAGCT ATTCAGAGCT ATTCGGAGCTATTCAGAGCT (mut2) ATTCAGAGCTATTCGGAGCT (mut1) Homo

37、allele：在同一基因locus中，同一突变位点（site）向不同方向发生突变所形成的等位基因 heteroallele：在同一基因locus中，不同突变位（site） 发生突变所形成的等位基因 site Gene locus Mut a1 Mut a2siteW.t AMut a1Mut a2ATTCTGAGCT ATTCGGAGCTATTCTGAGATATTCGGAGCTATTCGGAGAT (mut)ATTCTGAGATATTCTGAGCT (W.t) Lac. OperonLactose3.5 操纵子理论 (Lactose operon 1961. Jacob, Monod ) I

38、P O Z Y A zya生物体性状的表现往往具有上位性互作的遗传效应某一基因功能的表现是若干基因组成的信息表达的整体行为最古老的观点是将基因本身看作是生物的结构物质;第二种观点，认为基因是酶（或像酶一样起作用），作为体内化学过程的催化剂;基因被看成是能量传递的一种手段;把基因看作是特殊信息的传递者;现代基因阶段实际上是重新认识基因的阶段，基因是DNA分子中含有特定遗传信息的一段核苷酸序列，是遗传物质的最小功能单位。对于编码蛋白质的结构基因来说，基因是决定一条多肽链的DNA片段。 Transcritable, translatable gene ( Z,Y,A ) Transcritable

39、but non-translatable gene ( tDNA, rDNA ) Non-transcritable, non-translatable gene ( promoter, operator ) 3.6 基因的类型 Cis action elementAffects the activity only of DNA sequences on its own molecular of DNA, this property usually implies that the factor does not code for protein通过核苷酸自身的特异二级结构控制与它紧密连锁的结

40、构基因的表达。一般不编码蛋白质(无基因产物的DNA功能区)。 trans action factorAffects the activity of any gene located on genome by its translated product.通过扩散自身表达产物（酶，调节蛋白）控制其他基因的表达可转录，可翻译调节蛋白的DNA功能区可通过互补测验体系确定其功能区域4. The Nature of Genetic Material遗传物质的本质The material responsible for hereditary information must have three prin

41、cipal characteristics: It must contain, in a stable from, the information for an organisms cell structure, function, development, and reproduction.It must replicate accurately so that progeny cells have the same genetic information as the parental cell.It must be capable of change. Without change, o

42、rganisms would be incapable of variation and adaptation, and evolution could not occur.Hello, I am Friedrich Miescher （1844-1895）. I was interested in the chemistry of cells. In 1869, I began working with white blood cells.The studies that eventually revealed the chemistry of genes began in Tbingen,

43、Germany, in 1869.white blood cells are the major component of pus from infections. He collected a lot of pus from surgical bandages at local hospital.He used a salt solution to wash the pus of the bandages. When he added a weak alkaline solution to the cells, the cells lysed and nuclei precipitated

44、out of the solution. From the cell he isolated a unique chemical substance, which he called nuclein. Chemically, nuclein is high in phosphonus.Chromosomes are carriers of hereditary information: Chromosomes, the thread-like structure in the nucleus some exhibit a characteristic splitting behavior, i

45、n which each daughter cell formed by cell division receives an identical complement of chromosomes.The number of chromosomes in each cell differs from one biological species to the next, the number of chromosomes is nearly always constant within the cells of any one species.These made it seem likely

46、 that chromosomes were the carriers of the genes.Chemical analysis revealed that chromosomes are composed of protein and nucleic acid.DNA, the molecule that he worked on, has only four different units, called nucleotides. Tetranucleotide hypothesis.The amount of DNA per cell is constant, the amount

47、and kinds of protein and other large molecules differ greatly from one cell type to another.Phoebus Levene (1869-1940)Proteins are made up of 20 different subunitsaminos. Proteins come in many different shapes and sizes. The variety made proteins a good candidate as the molecules of heredity. DNA wa

48、s thought to be too simple a molecule to account for the variation found in living organisms, serving merely as a molecular scaffold 分子骨架4.1 Griffiths Transformation ExperimentAny researcher who hoped to demonstrate that DNA was the genetic material had a double handicap. Such experiments had to dem

49、onstrate not only that DNA is the genetic material but also that proteins are not the genetic material. The idea that genetic material is nucleic acid had its roots in the discovery of transformation in 1928, Frederick Griffith（弗雷德里克 格利菲斯）S strain is virulent polysaccharide capsule prevents detectio

50、n by hosts immune systemR strain is benign lacking a protective capsule, it is recognized and destroyed by hosts immune system？R strain is benignS strain is virulent killed S strain cells are benignlived R strain cells were transformed R strainLive S and R strains isolated from dead mouseThe heat-ki

51、lled virulent S bacteria converted live avirulentR cells into virulent S cells. Calling the phenomenon transformation.By 1931, Henry Dawson and his coworkers showed that transformation could in vitro.By 1933, Lionel J. Alloway had refined the in vitro experiments using extracts from S cells added to

52、 lived R cells. The soluble filtrate from the heat-killed S cells was as effective in inducing transformation as were the intact cells.Oswald Avery（奥斯瓦德 埃弗里）, Colin MacLeod（科林 麦克劳德）, and Maclyn McCarty（麦克林 麦卡迪） 4.2 DNA: The Transforming Material S cells in liquid culture mediumCentrifugeS cells spun

53、 to bottom of tubeHeat-killHomogenize cellsRecover S filtrateExtract carbohydrates, lipid,and proteinsTreat with proteaseTreat with ribonucleaseTreat with deoxyribonucleaseAssay for TransformationTreat with proteaseTreat with ribonucleaseTreat with deoxyribonucleaseAssay for TransformationR cells +S

54、 filtrateTransformation occursR cells +Protease-treated S filtrateTransformation occursR cells +RNase-treated S filtrateTransformation occursR cells +DNase-treated S filtrateNo transformation occursOnlyR cells Active factor is DNAR cells+S cellsActive factor is not RNAR cells+S cellsActive factor is

55、 not proteinR cells+S cellsS contains active factorUltracentrifugation：Spun the transforming substance in an ultracentrifuge to estimate its size. The material with transforming activity sedimented rapidly (moved rapidly toward the bottom of the centrifuge tube), suggesting a very high molecular wei

56、ght, characteristic of DNA.超速离心：利用超速离心机测定转化物质的大小。具有转化活性的物质沉降速度快，分子量大，具有DNA 的特征。Electrophoresis：Placed the transforming substance in an electric field to see how rapidly it moved. The transforming activity had a relatively high mobility, also characteristic of DNA because of its high charge-to-mass r

57、atio.电泳：通过凝胶电泳测定转化物质在电场中的迁移率，结果发现其迁移率较高，如同具有较高电荷/分子量比值的DNA 的电泳行为。Elementary Chemical Analysis：Yielded an average nitrogen-to-phosphorus ratio of 1.67, about what one would expect for DNA, which is rich in both elements, but vastly lower than the value expected for protein, which is rich in nitrogen

58、but poor in phosphorus. Even a slight protein contamination would have raised the nitrogen-to-phosphorus ratio.Ultraviolet Absorption Spectrophotometry：Placed a solution of the transforming substance in a spectrophotometer to see what kind of ultraviolet light it absorbed most strongly. Its absorpti

59、on spectrum matched that of DNA. That is, the light it absorbed most strongly had a wavelength of about 260 nanometers, in contrast to protein, which absorbs maximally at 280 nm.紫外吸收光谱学：利用分光光度计测定转化物质的紫外吸收，其吸收光谱与DNA的吸收光谱相吻合。即在260nm处具有强烈吸收峰，而蛋白质的吸收峰在280nm处。 Viral Genes Are Also Nucleic Acids Alfred He

60、rshey and Martha Chase，1952 Containing a DNA core and a protective shell built up by the aggregation of a number of different protein molecules.Phage attaches to host cell and injects DNAPhage DNAcircularize New phage DNA and proteins are synthesized and assembled into phagesCell lyses, releasing ph