動物分子遺傳學

1、動物分子遺傳學,馮登偵 寧夏大學農(nóng)學院,主要參考書,1 徐晉麟等．現(xiàn)代遺傳學原理．科學出版社， 2001 2 閻隆飛等．分子生物學．中國農(nóng)業(yè)大學出版社，19973 孫乃恩等．分子遺傳學．南京大學出版社，19964 解生勇．分子細胞遺傳學．中國農(nóng)業(yè)科技出版社，19985 李 寧等． 動物遺傳學．中國農(nóng)業(yè)出版社，20036 楊業(yè)華．分子遺傳學．中國農(nóng)業(yè)出版社，20017 李振剛．分子遺傳學．科學出版社，2000

2、8 朱玉賢等．現(xiàn)代分子生物學．高等教育出版社，19979 林 劍．免疫遺傳學．高等教育出版社，199710 LELAND H.HARTWELL,LEROY HOOD,MICHAEL L.GOLDBERG.遺傳學-從基因到基因組(GENETICS-from genes to genomes ).科學出版社.2003,遺傳學(genetics)的研究內容,All living organisms (from single –cell b

3、acteria 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，grow，reproduce，and survive in thei

4、r environments.,遺傳學(genetics)的研究內容,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 us

5、e it during their lifetime.,第一章 遺傳物質的基礎 一、遺傳物質,核酸是遺傳物質，存在于任何生命形式的有機體。DNA是最普遍的遺傳物質。在少數(shù)低等生物中RNA是遺傳物質。DNA中貯存了大量的遺傳信息。,DNA ----bearer of genetic information,For nearly 4 billion years the double-stranded DNA molecule

6、has served as the almost universal bearer of genetic information.3.7billion years ago the earliest bacterial cell incorporated it into their chromosomes .About 2 billion years ago, when the eukaryotic precursors of p

7、lants, 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

8、 which 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 li

9、ttle or no oxygen, DNA can withstand a wide range of temperature, pressure, and humidity and remain relative intact for hundreds, thousands, even millions of years. This ancient DNA still carries readable sequence . Co

10、mparisons 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 material,Chemical characterization localizes DNA

11、in the chromosomesBacterial transformation implicates DNA as the substance of genesExperiment of infecting bacterial cell with bacteriophages,Chemical characterization localizes DNA in the chromosomes,1869 friedrich ex

12、tracted a 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 .DNA is composed of four different subu

13、nit (nucleotides) linked in a long chain. The bonds joining one nucleotides to another are covalent phosphodiester bonds.,,Nitrogenous base,Sugar,Phosphate,,,,,nucleotides,phosphodiester bonds,,,,,Localizes DNA in the c

14、hromosomes,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 molecular has any

15、thing 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 po

16、tential for diversity and were better suited to serve as the genetic material. Scientists assumed that even though DNA was an important part of chromosomes structure, it was too simple to specify the complexity of gene

17、s.,Bacterial transformation implicates DNA as the substance of genes,Several studies dispelled the idea that DNA cannot be the genetic material .1928,Griffith published the astonishing finding that genetic information f

18、rom 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,Av

19、ery,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.1944,Avery and two coworker ,MacLeod and McCarty, p

20、ublished the cumulative findings of experiments designed to determine the transforming principle’s chemical composition.,,,Living R form,heart-killed S cellcomponents,,,,PurifiedTransformingprinciple,,,protease,,,,,

21、Protein destroyed,RNA destroyed,DNA destroyed,Fat e1iminated,indicates predominanceof DNA,RNase,DNase,Ultracen-trifugation,Physical andchemicalanalysis,,Introduce into R cells,S cell Transfor-mation,,,Introdu

22、ce into R cells,Introduce into R cells,Introduce into R cells,,,,S cell Transfor-mation,,S cell notTransfor-mation,S cell Transfor-mation,,,Experiment of infecting bacterial cell with bacteriophages,Convincing e

23、vidence that genes are DNA: The molecular carriers the information required for the replication of bacterial viruses. Hershey and Chase anticipated that they could assess the relative importance of DNA and protein in ge

24、ne transmission by infecting bacterial cells with viruses called phages, short for bacteriophages.,,,煙草花葉病毒感染,,三 最早的遺傳物質？,最早的“生命系統(tǒng)” 必須能夠復制與進化最早有生命的分子是核酸（堿基能夠互補進行復制）而不是蛋白質。第一個有生命的分子一定是RNA，而不是DNA：1 只發(fā)現(xiàn)有催化活性的RNA；2 RNA的核

25、糖環(huán)上具有2’-OH；3 核糖核苷二磷酸還原酶；4 在模擬的前生命系統(tǒng)中，核糖比脫氧核糖容易合成。,1 RNA它既是遺傳物質，又是酶。,,模板,,負鏈模板、正鏈酶,新鏈的合成,2 核糖環(huán)上的2’-OH,⑴可以使RNA折疊成高級結構,,RNA折疊成高級結構,2 核糖環(huán)上的2’-OH,⑵ 2’-OH使RNA具有催化活性,3 核糖核苷二磷酸酸還原酶Ribonucleotide diphosphate reductase,,四、

26、核酸之外的遺傳物質—朊病毒, ？？,朊病毒（ prion）：蛋白樣感染性粒子。PrP(PrPC)是朊病毒蛋白（ prion protein），一個在正常腦組織中表達的蛋白質，核基因編碼， 只含有?螺旋, 幾乎無?折疊，對蛋白酶敏感，沒有傳染性。病變的 PrP(PrPSc)N-端缺少67個氨基酸，含有約40%的?折疊 ，20%的?螺旋，對蛋白酶穩(wěn)定，有傳染性。,朊病毒,PrPSc本身不足以致病，只有進入細胞內或進入已表達PrP

27、c細胞時才有毒性。 感染是PrPc和PrPSc共同作用結果。PrPSc產(chǎn)生于內源性PrPc：感染性的PrPSc使內源性PrPc活化，使PrPc由無害結構轉變?yōu)榭筛腥镜腜rPSc的結構。羊的瘙癢?。⊿criapie）（導致山羊和綿羊退行性神經(jīng)疾病）和影響人腦功能的庫魯?。↘ulu，新幾內亞震顫?。┖涂?雅氏（Creuzfeld-Jakob Disease）及瘋牛病的致病因子都是朊病毒。,五、The evolution of biolo

28、gical 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 –processing molecule to appear

29、.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 pro

30、duce molecules capable of catalyzing the chemistry of 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

31、 storage and replication functions of RNA, while proteins ,with their far greater capacity for diversity, preempted the functions derived from RNA,s three –dimensional folding.The information contained in the sequence o

32、f 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 sequence o

33、f 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

34、molecular specialization.,The evolution of biological information,The evidence for the common origin of all living forms is present in their DNA sequence. All living organisms use essentially the same arbitrary genetic

35、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 bases in any organism,s DNA specifies .,All living things are clos

36、ely 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 protei

37、ns in bacteria ,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 th

38、ings are closely related,The close relatedness of all living organisms at the molecular level has great significance for an understanding of biology . It makes it possible to combine bits and pieces learned from differen

39、t organisms into a global understanding of molecular and cellular biology that is valid for all organisms. Even though controlled experimentation with human is usually impossible ,the relatedness of organisms allows us

40、 to learn about biology from mice, flies, worms, peas yeast, and other organisms that are accessible to experimentation.,,第二章 遺傳物質結構與特性,第一節(jié)、DNA的雙螺旋結構第二節(jié)、DNA的復性和變性第三節(jié)、DNA 的二級結構第四節(jié)、DNA的超螺旋結構,第一節(jié) DNA的雙螺旋結構,The Watson-Cr

41、ock model: DNA is a double helix. Nucleotides are the basic building blocks of DNA , a DNA chain composed of many nucleotides has polarity. At the end ,called the 5’end, the sugar of the terminal nucleotide has free 5

42、’end.At the other is 3’end of chain ,it is the 3’carbin of the final nucleotide that is free.A directional base sequence can carry information .The double helix contains two anti-parallel chain that associate by comple

43、mentary base pairing .,核糖,脫氧核糖,嘌呤： 腺嘌呤A、鳥嘌呤G,嘧啶： 胸腺嘧啶T 、 胞嘧啶C 、 尿嘧啶U,9-N,,1’-C,,1’-C,,,1-N,脫氧核苷,,核苷,,核苷,核苷酸,含氮堿+脫氧核糖→脫氧核苷 A,G,T,C +磷酸 脫氧核糖核

44、苷酸 DNA,,,Chargaff規(guī)律,A=T；G=C不對稱比率(A+T)/(G+C),脫氧核糖核甘酸,核糖核甘酸,,,,Twist 36°,C-G,T-A,,,,,8.5 Å,11.7 Å,7.5 Å,5.7 Å,DNA分子中：

45、,堿基：扁平；在分子內部；幾乎完全疏水糖-磷酸糖是扭曲的環(huán)；磷酸是四面體；在分子外部；親水；,DNA分子為什么能夠穩(wěn)定？,次級鍵的重要作用van de Waals力氫鍵疏水作用力離子鍵形成雙鏈,次級鍵,次級鍵的重要性不僅在于可以決定哪些原子依次相連，而且還能使柔性大分子（如多肽鏈、多核苷酸鏈）具有一定的空間構象。使一個分子結合與另一個分子；不會使分子在細胞內形成牢固的晶格；平均壽命不足1秒；形成與斷裂無需酶的

46、參與。,疏水作用（堿基堆積力）,疏水基團避開水相，相互聚集的作用。相對于堿基平面，是垂直方向上的作用力。堿基間的相互作用與堿基環(huán)的大小成正比：嘌呤-嘌呤＞嘌呤-嘧啶＞嘧啶-嘧啶,穩(wěn)定DNA雙螺旋的力：,堿基堆積力 氫鍵離子鍵,線性大分子的主鏈：,有規(guī)則的重復單位以相同的方向排列，如：肽鏈：-CONH多核苷酸鏈：3’，5’-磷酸二酯鍵可形成規(guī)則的次級鍵可形成穩(wěn)定的螺旋結構。,DNA是雙鏈,堿基在內：形成氫鍵避免與水接

47、觸糖-磷酸在外：與堿性蛋白質結合,,,DNA structure is the foundation of genetic function,The double helical structure of DNA provides a potential solution to the questions: how does the molecule carry information?How is that informati

48、on 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

49、 capacity 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 baseMuch of DNA’s sequence –specific informati

50、on is accessible only when the double helix is unwound.Some genetic information is accessible even in intact ,double –stranded DNA molecule. the major and minor grooves and in part from conformational irregularities in

51、 the sugar-phosphate backbone,第二節(jié) DNA的變性與復性,一 核酸的紫外吸收二 核酸的變性三 變性核酸的復性四 分子雜交,一 、紫外吸收,堿基、核苷、核苷酸以及核酸在紫外波段有強烈吸收，最大吸收值在260nm。在50μɡ/ml雙鏈DNA的260nm吸收值為1，單鏈的吸收值為1.37，游離堿基或核苷酸為1.6。,,二 核酸的變性,變性（Denaturation）或解鏈（melting）：DNA在緩

52、慢加熱時，氫鍵斷裂、雙鏈解開，產(chǎn)生單鏈的DNA分子。變性是爆發(fā)式的，在很窄的溫度范圍內，化學性質改變，沉降速度下降，紫外吸收增加（增色反應）。,溶解溫度 Tm,溶解溫度Tm:是DNA雙螺旋結構失去一半時的溫度(也就是DNA在Å2600紫外線吸收值達到最大值一半時的溫度)也稱DNA的熔點。在生理狀態(tài)下Tm值為85-95度。,影響Tm的因素,① DNA的均一性:均一性越高,變性的溫度越窄。②G-C含量:G-C含量與變性溫度成

53、正比。 Tm=69.3+0.41(G+C)%③介質中離子強度:強度越低,Tm越低,溫度范圍越窄,一般DNA的保存在1mol/LNaCl。④ DNA的結構:環(huán)性要高于線形。,,,,不同DNA結構條件,A-T,A-T,G-C,Helix Melting,Effect of [Salt] on Tm,三、核酸的復性,變性DNA在適當?shù)臈l件下，兩條彼此分開的鏈重新締合成為雙螺旋的過程稱為復性（Renaturation）或退火（Ann

54、ealing）。變性熱DNA驟然冷卻，不能復性。熱變性DNA緩慢冷卻（退火），可以復性。復性依賴于兩條互補專一性堿基配對。,,影響復性的因素,⑴ DNA的復雜性：結構簡單，容易配對，復性速度快；而復雜序列正確配對難度大，復性慢。⑵ DNA濃度：濃度高碰撞機會多，復性快。⑶DNA片段長度：片段越長，擴散速度慢，復性時間越長。⑷ 溫度：復性溫度比Tm低25度，溫度低，減少分子運動，復性慢。⑸離子強度：磷酸基團有排斥力，高鹽有利

55、復性。,,,,,,,四、核酸的分子雜交,分子雜交（Hybridization）：兩條來源不同，但具有互補序列的DNA單鏈（或DNA單鏈與RNA鏈）去掉變性條件后，能夠退火復性形成雙鏈DNA雜交分子（或DNA/RNA雜交分子）的過程。,分子雜交技術,分子雜交技術是利用DNA復性動力學原理用一條單鏈DNA或RNA與另一被檢測的DNA單鏈形成雙鏈來測定某特定序列是否存在。分子雜交技術必須有探針，探針是利用同位素或非同位素標記的短的特異性DN

56、A或RNA片段。原位分子雜交、斑點雜交、Southern雜交，Northern雜交，Western雜交，電鏡觀察,The “Hyperchromic Shift”,原位雜交,玻片原位雜交：分裂中期染色體或組織切片，在緩沖液中緩慢變性，加入探針，通過放射自顯影或染色，觀察基因在染色體上位置。用途：基因定位、組織中RNA分布、基因表達。膜上原位雜交：將菌落或噬菌斑印在尼龍膜（只吸附單鏈DNA）上，NaOH處理，用無關單鏈DNA預雜交，

57、經(jīng)中和后用同位素探針和膜放在緩沖液緩慢復性，放射自顯影篩選陽性克隆。用途：從基因文庫中釣基因。,,,斑點雜交（Dot Blotting）,又稱狹縫雜交：將總DNA直接滴在（通過長條狹縫印在）尼龍膜上，變性、預雜交、中和、洗脫、干燥，加入探針雜交。用途：測定生物是否含有特殊基因、序列來分析DNA樣品間的同源性。缺點：假陽性多。,Southern雜交,Southern 1975年建立的，DNA電泳后，將電泳條吸附到尼龍膜上，和DNA探

58、針雜交。用途：染色體的物理圖譜、限制性片段長度多態(tài)性、動物園印記（是一種動物的DNA探針和其他動物的DNA進行雜交）。,Northern雜交和Western雜交,Northern雜交：提取生物總RNA或mRNA，變性電泳，轉膜、用DNA雜交。用途：測定基因表達的時空性。Western雜交：蛋白質電泳后轉移到尼龍膜上，用抗體結合。用途：蛋白表達、蛋白活性等。,電鏡觀察,先進行雜交，在做電鏡觀察。異源雙鏈定位法（Heterdupl

59、ex Mapping）：環(huán)形不配對的區(qū)域可能是缺失或插入片段。R-環(huán)定位法（R—loop MaPPing）：標記的mRNA和待測DNA雙鏈雜交，檢測RNA位置。用途：基因定位、內含子測定、缺失或插入片段的發(fā)現(xiàn)。,第三節(jié)、DNA二級結構,The double helix may assume alternative formsB型DNA 、A型DNA （ 右旋）；Z型DNA（左旋）反向重復序列、DNA鏈的修飾三鏈和四鏈DNA

60、some DNA molecules are circular instead of linearSome 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雙螺旋結構模型，右手螺旋，每匝10個堿基對（溶液中10.5堿基對），在溶液中DNA一般為B型構象。,A-DNA（右旋）,當B-DNA脫水，或加入乙醇或鹽使水的活性降低可

61、轉變?yōu)锳-DNA。A-DNA使相鄰磷酸基間的距離縮短0.1nm，每匝11個堿基，螺距為2.8nm，堿基對向大溝移動0.5nm。A-DNA主要是DNA與RNA雜合體及RNA雙螺旋區(qū)的螺旋形式，或A-T豐富區(qū)。,Z-DNA（左旋）,當DNA鏈中連續(xù)出現(xiàn)鳥嘌呤和胞嘧啶二核苷酸d（CGCGCG）時，在這一區(qū)段內DNA形成Z型構象。Z-DNA中堿基不是對稱的位于螺旋軸附近而向邊緣延伸，使得大溝變淺，小溝變深變窄。在溶液中，鹽離子濃度低時d（C

62、GCGCG）以B-DNA存在，在高鹽離子濃度時以Z-DNA存在。胞嘧啶甲基化可使B-DNA轉變?yōu)閆-DNA。,A -DNA B -DNA Z-DNABp/圈 11 10.4 12旋轉/bp +34.7° +34.6° -30.0°上升nm /bp

63、 0.256 0.338 0.571上升nm /圈 2.8 3.4 6.9螺旋直徑nm 0.23 0.19 0.18,,Structural Variation Defined by Bases,,,normal,frequent,never,Never (except in inte

64、rcalation),Common,Common,,,PropellerTwist,,,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)

65、,A,A,B,Z,Z-DNA Phosphate Backbone is Kinked,反向重復序列,發(fā)針結構hairpin（莖環(huán)結構stem-loop）十字型結構cruciform,發(fā)針結構hairpin,當一條鏈上的一段序列與另一段序列互補且相離不遠時，單鏈就會自動折疊回來，形成了局部的雙鏈區(qū)，叫做莖（stem），莖的一端由不互補的序列形成一個環(huán)（loop），這種結構叫做發(fā)針（hairpin），或莖環(huán)（stem－loop）。

66、5’-GCTTTT……AAAAGC -3’,,,,,反向重復序列,如果雙鏈DNA含有結構，如： 5’-GCTTTT……AAAAGC -3’ CGAAAA……TTTTCG這段序列無論從哪個方向閱讀，兩條鏈的序列都是相同的。也就是形成了一個序列的兩個拷貝，兩個拷貝處于相反的方向，這樣的兩個拷貝叫做反向重復序列（inverted repeats）。,,兩個反向重復序列加在一起叫做回文結構（palindrome）?；匚?/p>

67、結構的正式名稱叫做雙重對稱區(qū)（region of dyalsymmetry），對稱軸將兩個反向重復序列分開。,回文結構palindrome,回文詩,賞花歸去馬如飛暮已時醒微力酒,賞花歸去馬如飛去馬如飛酒力微酒力微醒時已暮醒時已暮賞花歸,在雙螺旋DNA中，如果回文結構較長， 每個單鏈都有機會形成發(fā)針，這兩個相對的發(fā)針就形成了十字形結構（cruciform）。,十字形結（cruciform）。,,,,,,,DNA鏈的修飾,堿基的修飾

68、甲基化作用核苷酸序列對DNA鏈的修飾,,,完全甲基化→,半甲基化→,未甲基化→,,,,,三鏈和四鏈DNA,,,,,第四節(jié)、DNA的超螺旋與 拓撲異構酶,,DNA的超螺旋結構,閉合環(huán)狀DNA可以形成超螺旋結構supercoil正超螺旋（左旋）負超螺旋（右旋）,負超螺旋,當DNA分子繞著它的軸向反時針方向旋轉時（與右手螺旋相反的方向），就產(chǎn)生負超螺旋negative surpercoils。負超螺旋為右旋，約每2