动物分子传学ppt课件

1、 动物分子遗传学动物分子遗传学 冯登侦 宁夏大学农学院 主要参考书v1 1 徐晋麟等现代遗传学原理科学出版社，徐晋麟等现代遗传学原理科学出版社， 2001 2001 v2 2 阎隆飞等分子生物学中国农业大学出版社，阎隆飞等分子生物学中国农业大学出版社，19971997v3 3 孙乃恩等分子遗传学南京大学出版社，孙乃恩等分子遗传学南京大学出版社，19961996v4 4 解生勇分子细胞遗传学中国农业科技出版社，解生勇分子细胞遗传学中国农业科技出版社，19981998v5 5 李李 宁等宁等 动物遗传学中国农业出版社，动物遗传学中国农业出版社，20032003v6 6 杨业华分子遗传学中国农业出版

2、社，杨业华分子遗传学中国农业出版社，20012001v7 7 李振刚分子遗传学科学出版社，李振刚分子遗传学科学出版社，20002000v8 8 朱玉贤等现代分子生物学高等教育出版社，朱玉贤等现代分子生物学高等教育出版社，19971997v9 9 林林 剑免疫遗传学高等教育出版社，剑免疫遗传学高等教育出版社，19971997v10 leland h.hartwell,leroy hood,michael l.goldberg.10 leland h.hartwell,leroy hood,michael l.goldberg.遗传学遗传学- -从基因到基因组从基因到基因组(genetics-fr

3、om genes to (genetics-from genes to genomes ).genomes ).科学出版社科学出版社.2003.2003 遗传学(genetics)的研究内容vall 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 i

4、nformation to develop，grow，reproduce，and survive in their environments.遗传学(genetics)的研究内容vgenetics: the science of heredity ,is at its core the study of biological information. vgeneticists examine how organisms pass biological information on to their progeny and how they use it during their lifetim

5、e. 第一章 遗传物质的基础 一、遗传物质v核酸是遗传物质，存在于任何生命形式的有机体。vdna是最普遍的遗传物质。v在少数低等生物中rna是遗传物质。vdna中贮存了大量的遗传信息。dna -bearer of genetic information vfor nearly 4 billion years the double-stranded dna molecule has served as the almost universal bearer of genetic information.v3.7billion years ago the earliest bacterial ce

6、ll incorporated it into their chromosomes .vabout 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 vsince that time:vevolution has honed and expanded the sof

7、tware-the programs by which the molecular stores, transmits, and expresses genetic information.vthe hardware the structure of the dna molecular-has changed very little.dna -bearer of genetic informationvunder special conditions of little or no oxygen, dna can withstand a wide range of temperature, p

8、ressure, and humidity and remain relative intact for hundreds, thousands, even millions of years. vthis 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 designa

9、te dna as the genetic material vchemical characterization localizes dna in the chromosomesvbacterial transformation implicates dna as the substance of genesvexperiment of infecting bacterial cell with bacteriophageschemical characterization localizes dna in the chromosomesv1869 friedrich extracted a

10、 weakly acidic ( phosphorus -rich material )and named “nuclein”, its major component turned out to be dna- deoxyribonucleicacid , and it is found mainly in cell nuclei .vdna is composed of four different subunit (nucleotides) linked in a long chain. the bonds joining one nucleotides to another are c

11、ovalent phosphodiester bonds.nitrogenous basesugarphosphatenucleotidesphosphodiester bondslocalizes dna in the chromosomesvfeulgen reaction (chemical called schiff reagent ): which stained dna red .vfeulgen reaction shows that dna is localized chromosomes.vdna is a component of chromosomes does not

12、prove that the molecular has anything to do with genes. which is genetic material for dna and protein ?vbecause 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

13、serve as the genetic material. v 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 genesvseveral studies dispelled the idea that dna cannot be the g

14、enetic material .v1928,griffith published the astonishing finding that genetic information from dead bacterial cells somehow be transmitted to live cells .vthe ability of a substance to change the genetic characteristics of an organism is know as transformation.bacterial transformation is caused dna

15、v1931,avery,s laboratory 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.v1944,avery and two coworker ,macleod and mccarty, published the cumulative findings of experiments designed to determine th

16、e transforming principles chemical composition.living r form heart-killed s cellcomponents purifiedtransformingprincipleproteaseprotein destroyedrna destroyeddna destroyed fat e1iminatedindicates predominanceof dnarnasednaseultracen-trifugationphysical andchemicalanalysisintroduce into r cellss cell

17、 transfor-mationintroduce into r cellsintroduce into r cellsintroduce into r cellss cell transfor-mations cell nottransfor-mations cell transfor-mationexperiment of infecting bacterial cell with bacteriophagesvconvincing evidence that genes are dna: the molecular carriers the information required fo

18、r the replication of bacterial viruses. vhershey 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？v最早的“生命系统” 必须能够复制与进化v最早有生命的分子是核酸（碱基能够互补进行

19、复制）而不是蛋白质。v第一个有生命的分子一定是rna，而不是dna：v1 只发现有催化活性的rna；v2 rna的核糖环上具有2-oh；v3 核糖核苷二磷酸还原酶；v4 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。模板模板负链模板、正链酶负链模板、正链酶新链的合成2 核糖环上的2-ohvv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-ohv v2-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, ？v朊病毒（ prion）：蛋白样感染性粒

20、子。vprp(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。v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vprpsc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。vprpsc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。v羊的瘙痒病（scriapie）（导致山羊和绵羊退行性神经疾病）和

21、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。五五、the evolution of biological informationvthe evolution of biological information is a fascinating story spanning the 4.5 billion years of earth,s history.vmany biologists think that rna was the first information processing molecu

22、le 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 life .the evo

23、lution of biological informationvrna 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 derived from rna,s

24、 three dimensional folding.vthe information contained in the sequence of dna nucleotides specifies the sequence of amino acids in the proteins.the evolution of biological informationvwith division of labor, rna became an intermediary in converting the information in dna into the sequence of amino ac

25、ids in protein.vthe 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 informationvthe evidence for the com

26、mon origin of all living forms is present in their dna sequence. vall 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.vvia the code the order of bases in any org

27、anism,s dna specifies . all living things are closely related vthe 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 ,yeast ,plants

28、, worms, flies, mice, and human.vit 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 relatedvthe close relatedness of all living organisms at the molecular level has great signif

29、icance 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. veven though controlled experimentation with human is usually impossible ,the relat

30、edness 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v第一节、dna的双螺旋结构v第二节、dna的复性和变性v第三节、dna 的二级结构v第四节、dna的超螺旋结构第一节 dna的双螺旋结构vthe watson-crock model: dna is a double helix. vnucleotides are the

31、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.va directional base sequence can carry informati

32、on .vthe 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-n1-c1-c1-n脱氧核苷脱氧核苷核苷核苷核苷核苷核苷酸核苷酸含氮碱+脱氧核糖脱氧核苷 a,g,t,c +磷酸 脱氧核糖核苷酸 dnachargaff规律va=t；g=cv不对称比率(a+t)/(g+c)脱氧核糖核甘酸核糖核甘酸 twist 36c-gt-a 8.5 11.7 7.5 5.7 dna分子中

33、：v碱基：扁平；在分子内部；几乎完全疏水v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？v次级键的重要作用van de waals力氢键疏水作用力离子键v形成双链次级键v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。v使一个分子结合与另一个分子；v不会使分子在细胞内形成牢固的晶格；v平均寿命不足1秒；v形成与断裂无需酶的参与。疏水作用（碱基堆积力）v疏水基团避开水相，相互聚集的作用。v相对于碱基平面，是垂直方向上的作用力。v碱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

34、dna双螺旋的力：v碱基堆积力 v氢键v离子键线性大分子的主链：v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v可形成规则的次级键v可形成稳定的螺旋结构。dna是双链v碱基在内：形成氢键避免与水接触v糖-磷酸在外：与碱性蛋白质结合 dna structure is the foundation of genetic functionvthe double helical structure of dna provides a potential solution to the questions:v how does the molecule carry

35、 information?vhow is that information copied for transmission to generations?vwhat mechanisms allow the information to change ?vhow does the information govern the expression of phenotype?vthe double helical structure of dna endowing the molecule with the capacity to carry out all critical function

36、required of genetic material.dna structure is the foundation of genetic functionvdna stores information in the sequence of its basevmuch of dnas sequence specific information is accessible only when the double helix is unwound.vsome genetic information is accessible even in intact ,double stranded d

37、na 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v一 核酸的紫外吸收v二 核酸的变性v三 变性核酸的复性v四 分子杂交一 、紫外吸收v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。v在50/ml双链dna的260nm吸收值为1，单链的吸收值为1.37，游离碱基或核苷酸为1.6。 二 核酸的变性v变性（denaturation）或解链（melting）：dn

38、a在缓慢加热时，氢键断裂、双链解开，产生单链的dna分子。v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 tmv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。v在生理状态下tm值为85-95度。影响tm的因素v dna的均一性:均一性越高,变性的温度越窄。vg-c含量:g-c含量与变性温度成正比。v tm=69.3+0.41(g+c)%v介质中离子强度:强度越低,tm越低,温度范围越窄,一般dna的保存在1mol/lnacl。v dna的结构:环性要高于线形。

39、 不同不同dnadna结构条件结构条件a-ta-tg-chelix meltingeffect of salt on tm三、核酸的复性v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v 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。v dna浓度：浓度高碰撞机会多，复性快。vdna片段长度：片段越长，扩散速度慢，复性时间越长。v 温度：复性温度比tm低

40、25度，温度低，减少分子运动，复性慢。v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v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v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。v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片段。v原位分子杂交、斑点杂交、southern杂交，northern杂交，western

41、杂交，电镜观察the “hyperchromic shift”原位杂交v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。v用途：基因定位、组织中rna分布、基因表达。v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。v用途：从基因文库中钓基因。斑点杂交（dot blotting）v又称狭缝杂交：将总dna直接滴在（通过长条狭缝印在）尼龙膜上，变性、预杂交、中和、洗脱、干燥，加入探针杂交。v用途：测定生物是否含

42、有特殊基因、序列来分析dna样品间的同源性。v缺点：假阳性多。southern杂交vsouthern 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。v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vnorthern杂交：提取生物总rna或mrna，变性电泳，转膜、用dna杂交。v用途：测定基因表达的时空性。vwestern杂交：蛋白质电泳后转移到尼龙膜上，用抗体结合。v用途：蛋白表达、蛋白活性等。电镜观察v先进行杂交，在做电镜观察。v异源双链定位法（heterdu

43、plex mapping）：环形不配对的区域可能是缺失或插入片段。vr-环定位法（rloop mapping）：标记的mrna和待测dna双链杂交，检测rna位置。v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vthe double helix may assume alternative formsvb型dna 、a型dna （ 右旋）；z型dna（左旋）v反向重复序列、dna链的修饰v三链和四链dnav some dna molecules are circular instead of linearvsome viruses carry single stra

44、nded dnaabzhandednesspitchbase inclinationb型dna（右旋）vb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）v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）v当dna链中连续出现鸟嘌呤和胞嘧啶二核苷酸d（c

45、gcgcg）时，在这一区段内dna形成z型构象。vz-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。v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-dnabp/圈圈 11 10.4 12旋转旋转/bp +34.7 +34.6 -30.0上升上升nm /bp 0.256 0.338 0.571上升上升nm /圈圈 2.8 3.4 6.9螺旋直径螺旋直径nm 0.23 0.19 0.18structural variation defined

46、 by basesnormalfrequentnevernever (except in intercalation)commoncommonpropellertwistbucklereal lifesugar “pucker” conformationsa dnab dnab dnamajorminorz dnamajorminora dnamajorminorabzmimamamimimarotation about the n-glycosidic bondn3a,b dnaz dna (g only)aabzz-dna phosphate backbone is kinked反向重复序

47、列v发针结构hairpin（茎环结构stem-loop）v十字型结构cruciform发针结构hairpinv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）。v 5-gcttttaaaagc -3 v v 反向重复序列v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）。 v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