动物分子遗传学ppt课件.ppt

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1、动物分子遗传学,冯登侦 宁夏大学农学院,主要参考书,1 徐晋麟等现代遗传学原理科学出版社， 2001 2 阎隆飞等分子生物学中国农业大学出版社，1997 3 孙乃恩等分子遗传学南京大学出版社，1996 4 解生勇分子细胞遗传学中国农业科技出版社，1998 5 李 宁等 动物遗传学中国农业出版社，2003 6 杨业华分子遗传学中国农业出版社，2001 7 李振刚分子遗传学科学出版社，2000 8 朱玉贤等现代分子生物学高等教育出版社，1997 9 林 剑免疫遗传学高等教育出版社，1997 10 LELAND H.HARTWELL,LEROY HOOD,MICHAEL L.GOLDBERG.遗传学

2、-从基因到基因组(GENETICS-from genes to genomes ).科学出版社.2003,遗传学(genetics)的研究内容,All living organisms (from single cell bacteria and protozoa to multicellular plants and animal ) must store 、replicate 、transmit biological information to the next generation，and use vast quantities of information to develop，gr

3、ow，reproduce，and survive in their environments.,遗传学(genetics)的研究内容,Genetics: the science of heredity ,is at its core the study of biological information. Geneticists examine how organisms pass biological information on to their progeny and how they use it during their lifetime.,第一章 遗传物质的基础 一、遗传物质,核酸

4、是遗传物质，存在于任何生命形式的有机体。 DNA是最普遍的遗传物质。 在少数低等生物中RNA是遗传物质。 DNA中贮存了大量的遗传信息。,DNA -bearer of genetic information,For nearly 4 billion years the double-stranded DNA molecule has served as the almost universal bearer of genetic information. 3.7billion years ago the earliest bacterial cell incorporated it into

5、their chromosomes . About 2 billion years ago, when the eukaryotic precursors of plants, animals, and fungi evolved from simple cell, their chromosomes also carried a DNA molecular .,DNA -bearer of genetic information,Since that time: Evolution has honed and expanded the software-the programs by whi

6、ch the molecular stores, transmits, and expresses genetic information. The hardware the structure of the DNA molecular-has changed very little.,DNA -bearer of genetic information,Under special conditions of little or no oxygen, DNA can withstand a wide range of temperature, pressure, and humidity an

7、d remain relative intact for hundreds, thousands, even millions of years. This ancient DNA still carries readable sequence . Comparisons with equivalent expanses of modern DNA make it possible to identify the precise mutations that have fueled evolution .,二、Experiment designate DNA as the genetic ma

8、terial,Chemical characterization localizes DNA in the chromosomes Bacterial transformation implicates DNA as the substance of genes Experiment of infecting bacterial cell with bacteriophages,Chemical characterization localizes DNA in the chromosomes,1869 friedrich extracted a weakly acidic ( phospho

9、rus -rich material )and named “nuclein”, its major component turned out to be DNA- deoxyribonucleicacid , and it is found mainly in cell nuclei . DNA is composed of four different subunit (nucleotides) linked in a long chain. The bonds joining one nucleotides to another are covalent phosphodiester b

10、onds.,Nitrogenous base,Sugar,Phosphate,nucleotides,phosphodiester bonds,Localizes DNA in the chromosomes,Feulgen reaction (chemical called Schiff reagent ): which stained DNA red . Feulgen reaction shows that DNA is localized chromosomes. DNA is a component of chromosomes does not prove that the mol

11、ecular has anything to do with genes.,Which is genetic material for DNA and protein ?,Because proteins are built of different amino acids whereas DNA carries just four different subunit ,many researcher thought proteins had greater potential for diversity and were better suited to serve as the genet

12、ic material. Scientists assumed that even though DNA was an important part of chromosomes structure, it was too simple to specify the complexity of genes.,Bacterial transformation implicates DNA as the substance of genes,Several studies dispelled the idea that DNA cannot be the genetic material . 19

13、28,Griffith published the astonishing finding that genetic information from dead bacterial cells somehow be transmitted to live cells . The ability of a substance to change the genetic characteristics of an organism is know as transformation.,Bacterial transformation is caused DNA,1931,Avery,s labor

14、atory achieved transformation without using any animals at all, simply by growing R-form bacterial on medium in the presence of components from dead S forms. 1944,Avery and two coworker ,MacLeod and McCarty, published the cumulative findings of experiments designed to determine the transforming prin

15、ciples chemical composition.,Living R form,heart-killed S cell components,Purified Transforming principle,protease,Protein destroyed,RNA destroyed,DNA destroyed,Fat e1iminated,indicates predominance of DNA,RNase,DNase,Ultracen -trifugation,Physical and chemical analysis,Introduce into R cells,S cell

16、 Transfor- mation,Introduce into R cells,Introduce into R cells,Introduce into R cells,S cell Transfor- mation,S cell not Transfor- mation,S cell Transfor- mation,Experiment of infecting bacterial cell with bacteriophages,Convincing evidence that genes are DNA: The molecular carriers the information

17、 required for the replication of bacterial viruses. Hershey and Chase anticipated that they could assess the relative importance of DNA and protein in gene transmission by infecting bacterial cells with viruses called phages, short for bacteriophages.,烟草花叶病毒感染,三 最早的遗传物质？,最早的“生命系统” 必须能够复制与进化 最早有生命的分子

18、是核酸（碱基能够互补进行复制）而不是蛋白质。 第一个有生命的分子一定是RNA，而不是DNA： 1 只发现有催化活性的RNA； 2 RNA的核糖环上具有2-OH； 3 核糖核苷二磷酸还原酶； 4 在模拟的前生命系统中，核糖比脱氧核糖容易合成。,1 RNA它既是遗传物质，又是酶。,模板,负链模板、正链酶,新链的合成,2 核糖环上的2-OH, 可以使RNA折叠成高级结构,RNA折叠成高级结构,2 核糖环上的2-OH, 2-OH使RNA具有催化活性,3 核糖核苷二磷酸酸还原酶 Ribonucleotide diphosphate reductase,四、核酸之外的遗传物质 朊病毒, ？,朊病毒（ pr

19、ion）：蛋白样感染性粒子。 PrP(PrPC)是朊病毒蛋白（ prion protein），一个在正常脑组织中表达的蛋白质，核基因编码， 只含有螺旋, 几乎无折叠，对蛋白酶敏感，没有传染性。 病变的 PrP(PrPSc)N-端缺少67个氨基酸，含有约40%的折叠 ，20%的螺旋，对蛋白酶稳定，有传染性。,朊病毒,PrPSc本身不足以致病，只有进入细胞内或进入已表达PrPc细胞时才有毒性。 感染是PrPc和PrPSc共同作用结果。 PrPSc产生于内源性PrPc：感染性的PrPSc使内源性PrPc活化，使PrPc由无害结构转变为可感染的PrPSc的结构。 羊的瘙痒病（Scriapie）（导致山

20、羊和绵羊退行性神经疾病）和影响人脑功能的库鲁病（Kulu，新几内亚震颤病）和克-雅氏（Creuzfeld-Jakob Disease）及疯牛病的致病因子都是朊病毒。,五、The evolution of biological information,The evolution of biological information is a fascinating story spanning the 4.5 billion years of earth,s history. Many biologists think that RNA was the first information proc

21、essing molecule to appear .RNA molecules are very similar to DNA and are also composed of four subunits. Like DNA , RNA has the capacity to store ,replicate, mutate, and express information; like proteins , RNA can fold in three dimensions to produce molecules capable of catalyzing the chemistry of

22、life .,The evolution of biological information,RNA molecules are intrinsically unstable .Thus it is probable that the more stable DNA took over the linear information storage and replication functions of RNA, while proteins ,with their far greater capacity for diversity, preempted the functions deri

23、ved from RNA,s three dimensional folding. The information contained in the sequence of DNA nucleotides specifies the sequence of amino acids in the proteins.,The evolution of biological information,With division of labor, RNA became an intermediary in converting the information in DNA into the seque

24、nce of amino acids in protein. The separation that placed information storage in DNA and biological function in protein was so successful that all organism alive today descend from the first organisms that happened upon this molecular specialization.,The evolution of biological information,The evide

25、nce for the common origin of all living forms is present in their DNA sequence. All living organisms use essentially the same arbitrary genetic code in which various groupings of the 4 letters of the DNA and RNA alphabets encode the 20 letters of the amino-acid alphabet. Via the code the order of ba

26、ses in any organism,s DNA specifies .,All living things are closely related,The relatedness of all living organisms is also evident from comparisons of genes with similar functions in very different organisms. For example, there is striking similarity between the gene for many proteins in bacteria ,

27、yeast ,plants, worms, flies, mice, and human. It is possible to replace a gene from an organism into the genome of a very different organism and see it function normally in new environment .,All living things are closely related,The close relatedness of all living organisms at the molecular level ha

28、s great significance for an understanding of biology . It makes it possible to combine bits and pieces learned from different organisms into a global understanding of molecular and cellular biology that is valid for all organisms. Even though controlled experimentation with human is usually impossib

29、le ,the relatedness of organisms allows us to learn about biology from mice, flies, worms, peas yeast, and other organisms that are accessible to experimentation.,第二章 遗传物质结构与特性,第一节、DNA的双螺旋结构 第二节、DNA的复性和变性 第三节、DNA 的二级结构 第四节、DNA的超螺旋结构,第一节 DNA的双螺旋结构,The Watson-Crock model: DNA is a double helix. Nucleo

30、tides are the basic building blocks of DNA , a DNA chain composed of many nucleotides has polarity. At the end ,called the 5end, the sugar of the terminal nucleotide has free 5end.At the other is 3end of chain ,it is the 3carbin of the final nucleotide that is free. A directional base sequence can c

31、arry information . The double helix contains two anti-parallel chain that associate by complementary base pairing .,核糖,脱氧核糖,嘌呤： 腺嘌呤A、鸟嘌呤G,嘧啶： 胸腺嘧啶T 、 胞嘧啶C 、 尿嘧啶U,9-N,1-C,1-C,1-N,脱氧核苷,核苷,核苷,核苷酸,含氮碱+脱氧核糖脱氧核苷 A,G,T,C +磷酸 脱氧核糖核苷酸 DNA,Chargaff规律,A=T；G=C 不对称比率(A+T)/(G+C),脱氧核糖核甘酸,核糖核甘酸,Twist 36,C-G,T-A,8.5

32、 ,11.7 ,7.5 ,5.7 ,DNA分子中：,碱基： 扁平； 在分子内部；几乎完全疏水 糖-磷酸 糖是扭曲的环；磷酸是四面体； 在分子外部；亲水；,DNA分子为什么能够稳定？,次级键的重要作用 van de Waals力 氢键 疏水作用力 离子键 形成双链,次级键,次级键的重要性不仅在于可以决定哪些原子依次相连，而且还能使柔性大分子（如多肽链、多核苷酸链）具有一定的空间构象。 使一个分子结合与另一个分子； 不会使分子在细胞内形成牢固的晶格； 平均寿命不足1秒； 形成与断裂无需酶的参与。,疏水作用（碱基堆积力）,疏水基团避开水相，相互聚集的作用。 相对于碱基平面，是垂直方向上的作用力。 碱

33、基间的相互作用与碱基环的大小成正比： 嘌呤-嘌呤嘌呤-嘧啶嘧啶-嘧啶,稳定DNA双螺旋的力：,碱基堆积力 氢键 离子键,线性大分子的主链：,有规则的重复单位以相同的方向排列，如： 肽链：-CONH 多核苷酸链：3，5-磷酸二酯键 可形成规则的次级键 可形成稳定的螺旋结构。,DNA是双链,碱基在内： 形成氢键 避免与水接触 糖-磷酸在外： 与碱性蛋白质结合,DNA structure is the foundation of genetic function,The double helical structure of DNA provides a potential solution to

34、the questions: how does the molecule carry information? How is that information copied for transmission to generations? What mechanisms allow the information to change ? How does the information govern the expression of phenotype? The double helical structure of DNA endowing the molecule with the ca

35、pacity to carry out all critical function required of genetic material.,DNA structure is the foundation of genetic function,DNA stores information in the sequence of its base Much of DNAs sequence specific information is accessible only when the double helix is unwound. Some genetic information is a

36、ccessible even in intact ,double stranded DNA molecule. the major and minor grooves and in part from conformational irregularities in the sugar-phosphate backbone,第二节 DNA的变性与复性,一 核酸的紫外吸收 二 核酸的变性 三 变性核酸的复性 四 分子杂交,一 、紫外吸收,碱基、核苷、核苷酸以及核酸在紫外波段有强烈吸收，最大吸收值在260nm。 在50/ml双链DNA的260nm吸收值为1，单链的吸收值为1.37，游离碱基或核苷酸

37、为1.6。,二 核酸的变性,变性（Denaturation）或解链（melting）：DNA在缓慢加热时，氢键断裂、双链解开，产生单链的DNA分子。 变性是爆发式的，在很窄的温度范围内，化学性质改变，沉降速度下降，紫外吸收增加（增色反应）。,溶解温度 Tm,溶解温度Tm:是DNA双螺旋结构失去一半时的温度(也就是DNA在2600紫外线吸收值达到最大值一半时的温度)也称DNA的熔点。 在生理状态下Tm值为85-95度。,影响Tm的因素, DNA的均一性:均一性越高,变性的温度越窄。 G-C含量:G-C含量与变性温度成正比。 Tm=69.3+0.41(G+C)% 介质中离子强度:强度越低,Tm越低

38、,温度范围越窄,一般DNA的保存在1mol/LNaCl。 DNA的结构:环性要高于线形。,不同DNA结构条件,A-T,A-T,G-C,Helix Melting,Effect of Salt on Tm,三、核酸的复性,变性DNA在适当的条件下，两条彼此分开的链重新缔合成为双螺旋的过程称为复性（Renaturation）或退火（Annealing）。 变性热DNA骤然冷却，不能复性。 热变性DNA缓慢冷却（退火），可以复性。 复性依赖于两条互补专一性碱基配对。,影响复性的因素, DNA的复杂性：结构简单，容易配对，复性速度快；而复杂序列正确配对难度大，复性慢。 DNA浓度：浓度高碰撞机会多，复

39、性快。 DNA片段长度：片段越长，扩散速度慢，复性时间越长。 温度：复性温度比Tm低25度，温度低，减少分子运动，复性慢。 离子强度：磷酸基团有排斥力，高盐有利复性。,四、核酸的分子杂交,分子杂交（Hybridization）：两条来源不同，但具有互补序列的DNA单链（或DNA单链与RNA链）去掉变性条件后，能够退火复性形成双链DNA杂交分子（或DNA/RNA杂交分子）的过程。,分子杂交技术,分子杂交技术是利用DNA复性动力学原理用一条单链DNA或RNA与另一被检测的DNA单链形成双链来测定某特定序列是否存在。 分子杂交技术必须有探针，探针是利用同位素或非同位素标记的短的特异性DNA或RNA片

40、段。 原位分子杂交、斑点杂交、Southern杂交，Northern杂交，Western杂交，电镜观察,The “Hyperchromic Shift”,原位杂交,玻片原位杂交：分裂中期染色体或组织切片，在缓冲液中缓慢变性，加入探针，通过放射自显影或染色，观察基因在染色体上位置。 用途：基因定位、组织中RNA分布、基因表达。 膜上原位杂交：将菌落或噬菌斑印在尼龙膜（只吸附单链DNA）上，NaOH处理，用无关单链DNA预杂交，经中和后用同位素探针和膜放在缓冲液缓慢复性，放射自显影筛选阳性克隆。 用途：从基因文库中钓基因。,斑点杂交（Dot Blotting）,又称狭缝杂交：将总DNA直接滴在（通

41、过长条狭缝印在）尼龙膜上，变性、预杂交、中和、洗脱、干燥，加入探针杂交。 用途：测定生物是否含有特殊基因、序列来分析DNA样品间的同源性。 缺点：假阳性多。,Southern杂交,Southern 1975年建立的，DNA电泳后，将电泳条吸附到尼龙膜上，和DNA探针杂交。 用途：染色体的物理图谱、限制性片段长度多态性、动物园印记（是一种动物的DNA探针和其他动物的DNA进行杂交）。,Northern杂交和Western杂交,Northern杂交：提取生物总RNA或mRNA，变性电泳，转膜、用DNA杂交。 用途：测定基因表达的时空性。 Western杂交：蛋白质电泳后转移到尼龙膜上，用抗体结合。

42、 用途：蛋白表达、蛋白活性等。,电镜观察,先进行杂交，在做电镜观察。 异源双链定位法（Heterduplex Mapping）：环形不配对的区域可能是缺失或插入片段。 R-环定位法（Rloop MaPPing）：标记的mRNA和待测DNA双链杂交，检测RNA位置。 用途：基因定位、内含子测定、缺失或插入片段的发现。,第三节、DNA二级结构,The double helix may assume alternative forms B型DNA 、A型DNA （ 右旋）；Z型DNA（左旋） 反向重复序列、DNA链的修饰 三链和四链DNA some DNA molecules are circula

43、r instead of linear Some viruses carry single stranded DNA,A,B,Z,B型DNA（右旋）,B型DNA（右旋）是Wateson和Crick提出DNA双螺旋结构模型，右手螺旋，每匝10个碱基对（溶液中10.5碱基对），在溶液中DNA一般为B型构象。,A-DNA（右旋）,当B-DNA脱水，或加入乙醇或盐使水的活性降低可转变为A-DNA。A-DNA使相邻磷酸基间的距离缩短0.1nm，每匝11个碱基，螺距为2.8nm，碱基对向大沟移动0.5nm。A-DNA主要是DNA与RNA杂合体及RNA双螺旋区的螺旋形式，或A-T丰富区。,Z-DNA（左旋）

44、,当DNA链中连续出现鸟嘌呤和胞嘧啶二核苷酸d（CGCGCG）时，在这一区段内DNA形成Z型构象。 Z-DNA中碱基不是对称的位于螺旋轴附近而向边缘延伸，使得大沟变浅，小沟变深变窄。 在溶液中，盐离子浓度低时d（CGCGCG）以B-DNA存在，在高盐离子浓度时以Z-DNA存在。胞嘧啶甲基化可使B-DNA转变为Z-DNA。,A -DNA B -DNA Z-DNA Bp/圈 11 10.4 12 旋转/bp +34.7 +34.6 -30.0 上升nm /bp 0.256 0.338 0.571 上升nm /圈 2.8 3.4 6.9 螺旋直径nm 0.23 0.19 0.18,Structura

45、l Variation Defined by Bases,normal,frequent,never,Never (except in intercalation),Common,Common,Propeller Twist,Buckle,Real Life,Sugar “Pucker” Conformations,A DNA,B DNA,A DNA,Major,Minor,A,B,Z,Mi,Ma,Ma,Mi,Mi,Ma,Rotation About the N-Glycosidic Bond,N3,A,B DNA,Z DNA (G only),A,A,B,Z,Z-DNA Phosphate

46、Backbone is Kinked,反向重复序列,发针结构hairpin（茎环结构stem-loop） 十字型结构cruciform,发针结构hairpin,当一条链上的一段序列与另一段序列互补且相离不远时，单链就会自动折叠回来，形成了局部的双链区，叫做茎（stem），茎的一端由不互补的序列形成一个环（loop），这种结构叫做发针（hairpin），或茎环（stemloop）。 5-GCTTTTAAAAGC -3,反向重复序列,如果双链DNA含有结构，如： 5-GCTTTTAAAAGC -3 CGAAAATTTTCG 这段序列无论从哪个方向阅读，两条链的序列都 是相同的。也就是形成了一个序列

47、的两个拷贝， 两个拷贝处于相反的方向，这样的两个拷贝叫做 反向重复序列（inverted repeats）。,两个反向重复序列加在一起叫做回文结构（palindrome）。回文结构的正式名称叫做双重对称区（region of dyalsymmetry），对称轴将两个反向重复序列分开。,回文结构palindrome,回文诗,赏花归去马如飞 暮已时醒微力酒,赏花归去马如飞 去马如飞酒力微 酒力微醒时已暮 醒时已暮赏花归,在双螺旋DNA中，如果回文结构较长， 每个单链都有机会形成发针，这两个相对的发针就形成了十字形结构（cruciform）。,十字形结（cruciform）。,DNA链的修饰,碱基的

48、修饰 甲基化作用 核苷酸序列对DNA链的修饰,完全甲基化,半甲基化,未甲基化,三链和四链DNA,第四节、DNA的超螺旋与 拓扑异构酶,DNA的超螺旋结构,闭合环状DNA可以形成超螺旋结构supercoil 正超螺旋（左旋） 负超螺旋（右旋）,负超螺旋,当DNA分子绕着它的轴向反时针方向旋转时（与右手螺旋相反的方向），就产生负超螺旋negative surpercoils。 负超螺旋为右旋，约每200bp存在一个负超螺旋。,如果DNA旋转方向与分子本身缠绕方向相同，就在结构中产生正超螺旋（positive supercoils）。 正超螺旋使分子缠绕更紧密，为左旋。,A0267501,正超螺旋,“松弛” relaxed,如果一个DNA分子没有超螺旋，无论是封闭的还是开放的，都叫做“松弛” relaxed。,DNA Unwinding Causes Topological Problems,(Transcription),Unwound Parental Duplex,Over- Wound region,More Topological Problems,连接数（linking number, L） 盘绕数（twisting number, T） 超螺旋数（writhing number, W）,L= T + W,假设DNA分子放在一个平面上，闭环分子中一条多核苷