基因工程分子生物学第三章载体课件

1、 第三章 载体 第一节 载体的概念一、基因复制的基本单位复制子（replicon） The unit of DNA in which an individual act of replication occurs is called the replicon. Each replicon fires once and only once in each cell cycle. The replicon is defined by its possession of the control elements needed for replication. It has an origin at

2、which replication is initiated. It may also have a terminus at which replication stops (Jacob, Brenner, and Cuzin, 1963). 某一复制行为发生所在的DNA单位称为复制子(replicon)。 每个复制子在每个细胞周期中只“引发”一次。复制子是由它具有复制作用所必需的各种控制元件而定义的。它有一个复制启动的起点(origin)，可能还有一个复制终止的终点(terminus)。 Any sequence attached to an originor, more precisely

3、, not separated from an origin by a terminusis replicated as part of that replicon. The origin is a cis-acting site, able to affect only that molecule of DNA on which it resides 连在起点后的任何序列(或更确切地说，没有被终点从起点上分隔开的序列)均作为这个复制子的一部分进行复制。起点是一个顺式作用部位，只能对与它相连的DNA分子起作用。 A genome in a prokaryotic cell constitute

4、s a single replicon; 原核细胞内的一个基因组由单一复制子构成。 Bacteria may contain additional genetic information in the form of plasmids. A plasmid is an autonomous circular DNA genome that constitutes a separate replicon. A plasmid replicon may show single copy control, which means that it replicates once every time

5、the bacterial chromosome replicates. Or it may be under multicopy control, when it is present in a greater number of copies than the bacterial chromosome. 细菌可含有以质粒形式存在的附加遗传信息。 质粒是一种自主的环状DNA基因组，它含有一个与染色体分离的复制子。 质粒可以是单拷贝控制（single copy control）的，即，每次细菌染色体复制时它复制一次。也可以是多拷贝控制（single copy control）之下的，相对于细菌

6、染色体，它存在大量拷贝。 Each phage or virus DNA also constitutes a replicon, able to initiate many times during an infectious cycle. Perhaps a better way to view the prokaryotic replicon, therefore, is to reverse the definition: any DNA molecule that contains an origin can be replicated autonomously in the cel

7、l. 每个噬菌体或病毒也包含一个复制子， 但每次侵染周期能够起始多次复制。 所以，对于原核复制子来说：任何含有一个复制起点的DNA分子都可以在细胞内自主复制。 Each eukaryotic chromosome contains a large number of replicons. 每个真核染色体包含大量复制子。（The original formulation of the replicon in prokaryotes viewed it as a unit possessing both the origin and the gene coding for the regulato

8、r protein. Now, however, replicon is usually applied to eukaryotic chromosomes to describe a unit of replication that contains an origin; trans-acting regulator protein(s) may be coded elsewhere.)Cloning of E. coli oriC using genetic selection for ampicilin resistance and replication. Ampicilin-sens

9、itive (Amps) E. coli were transformed with (A) a collection of circularized EcoRI fragments of E. coli DNA (but no ampicilin-resistance gene is contained on any of the fragments), with (B) a circularized EcoRI fragment containing an ampicilin-resistance gene (but this fragment does not contain an or

10、igin of replication), or with (C) a collection of circularized molecules formed by joining the EcoRI fragment containing the ampicilin-resistance gene to each of the E. coli EcoRI fragments (one of which contains oriC). (A) and (B) do not give rise to ampicilin-resistant colonies. (C) gives rise to

11、a few ampicilin-resistant (Ampr) colonies, and each of the cells in each of these colonies contains an identical plasmid: the plasmids all have the 6.8 kb EcoRI fragment containing the ampicilin-resistance gene and a 9.0 kb EcoRI fragment containing oriC. pBR322的一个特性是它的复制依赖于宿主细胞的DNA聚合酶 I ，因此在一个温度敏感的

12、聚合酶突变型 polAts （ polA： DNA聚合酶I的编码基因, ts: temperature sensitive 42下不可复制）E.coli 中这一嵌合质粒得以在37和42中复制，但是如果这一嵌合质粒中的染色体复制起点丧失了功能，那么它便只能在37中复制，在42中便将由于不能复制而消失。pBR322带有抗药性基因APR，所以只要在42中测定细菌能否在含有氨苄青霉素的培养基上生长，便可以知道染色体复制起点的功能是否丧失。FIG. Consensus sequence of the minimal origin of the bacterial chromosome. The cons

13、ensus sequence is derived from six bacterial origin sequences, those of E. coli (2, 3), S. typhimurium (4), Enterobacter aerogenes (5), K. pneumoniae (5), Erwinia carotovora (6), and V. harveyi. The alignment of the six sequences is such that the least number of changes are introduced into the conse

14、nsus sequence. The two bases inserted (at positions 239 and240) anddeleted (at positions 246 and247) are counted as tworather thanfourchanges for calculations in the text. Inthe consensus sequence, a large capital letter means that the same nucleotide is found in all six origins; a small capital let

15、ter means the nucleotide is present in five of the six sequences; a lowercase letter is used when that nucleotide is present in three or four of six bacterial origins but only two different nucleotides are found at that site; and where three or four of the four possible nucleotides, or two different

16、 nucleotides plus a deletion, are found at a site, the letter nis used. Boldlarge capital letters locate positions 149, 242, and267, where single-base substitutions produce anoriC- phenotype inE. coli (31). The arrow at position 166 represents the first ribonucleotide of aRNAtranscribed byRNApolymer

17、ase in vitro (32). Thearrowsending between positions 71 and 72 and positions 107 and 108 mark the major RNA-DNAtransition sites (33). The directions of these three arrows show the direction of transcription. In the individual origin sequences, - means a deletion relative to the consensus sequence is

18、 present and . indicates that the nucleotide in the bacterial sequence is the same as the nucleotide in the consensus sequence. G-A-T-C sites are underlined in the consensus sequence andcertainE. coli restriction sites are noted. The minimal origin ofE. coli (34) is enclosed withinthe box. Thenumber

19、ingof the nucleotide positions is that used for E. coli (2, 3), and the upper left end is the 5 end. The four related 9-bp repeats, R1, R2, R3, and R4, are indicated by the arrows, with the 5 to 3 sequence given below the arrow.Figure Schematic of DNA polymerase III holoenzyme. DNA polymerase III ho

20、loenzyme contains 10 different subunits; arrows identify each of them. The three functional components of the holoenzyme are shown to the right of the diagram clamp (), core polymerase (), complex () The subunit dimer acts as a glue to bind together two core polymerases and one complex to form the P

21、ol III* subassembly (the holoenzyme lacking ) as shown in the diagram at the far right the clamp-loading complexThe clamp-loading complex consists of the delta () (38.7 kD) and delta () prime (36.9 kD), chi () (16.6 kD) and psi () (15.2 kD) subunits and either or both of the gamma () subunit (68.4 k

22、D) and the tau () subunit (71.1 kD). Both the gamma and tau subunits are encoded by the dnaX gene. This gene is translated in two different ways. If the gene is translated completely then the tau () subunit is synthesized. If translation slips or frameshifts part way through the reading frame then a

23、 stop codon is encountered, and the gamma () subunit is synthesized.Both the gamma () subunit and the tau() subunit are motor ATPases.The structures of many of these subunits have now been solved and they are beginning to reveal how the different subunits interact and function in the building of a r

24、eplisome and in its ongoing function.The subunit binds to the subunit and, in concert with the andsubunits and with ATP, it catalyses the opening of the dimer to permit passage of DNA. 四、载体的概念 基因克隆是要把一个外源基因导人寄主细胞，并使它得到扩增。然而，外源基因一般不具有复制起点，不是一个独立的复制子。 同时，对于寄主来说它又是外源的，会受到限制。所以一段外源基因进入寄主细胞后，很难生存和繁殖。为了使外

25、源基因能够生存和繁殖，可以将它与寄主内的独立复制子重组在一起，然后导入到寄主细胞。由于它被寄主内的独立复制子的携带和保护而生存和繁殖下来。这种携带和保护外源基因在寄主细胞内生存和复制的独立复制子被称为载体(vector)。 作为载体应具备的条件：（1）是一个独立的复制子，有复制起点；（2）有选择遗传标记；（3）具有多种选择的克隆位点。克隆位点为 RE位点，作为克隆位点的限制酶位点在一种载体出现的次数最多不超过两次。 质粒是亚细胞有机体。它的结构比病毒还要简单些，既没有蛋白质外壳，也没有细胞外的生命周期，只能够在寄主细胞内独立地增殖，并随着寄主细胞的分裂而被遗传下去。 1 质粒的生物学 在大肠杆

26、菌的各种菌株中，找到了许多种不同类型的质粒，其中已经作了比较详尽研究的主要有F质粒、R质粒和Col质粒。 F质粒 （ F : fertility） 又叫F因子或性质粒( sex plasmid)。它们能够使寄主染色体上的基因和F质粒一道转移到原先不存在该质粒的受体细胞中去。 R质粒 通称抗药性因子。它们编码有一种或数种抗菌素抗性基因，并且通常能够将此种抗性转移到缺乏该质粒的适宜的受体细胞，使后者也获得同样的抗菌素抗性能力。 Col质粒 即所谓产生大肠杆菌素因子，它们编码有控制大肠杆菌素合成的基因。大肠杆菌素是一类可以使不带有Col质粒的亲缘关系密切的细菌菌株致死的蛋白质。 天然质粒和 人工构建

27、的实验室质粒 质粒DNA covalently closed circles or CCC DNA open circles or OC DNA linearized plasmid Linear plasmid Fig. Effect of intercalation of ethidium bromide on supercoiling of DNA. As the amount of intercalated ethidium bromide increases, the double helix untwists, with the result that the supercoili

28、ng decreases until the open form of the circular molecule is produced. Further intercalation introduces excess turns in the double helix, resulting in supercoiling in the opposite sense (note the direction of coiling at B and D). For clarity, only a single line represents the double helix. 根据质粒能否在细胞

29、间进行传递，可把质粒分为自身传递性质粒(Selftransmissible conjugative Pasmid )和非自身传递性质粒。Transfer begins from oriT which is nicked by TraY/TraI complex at a nic site. F-plasmidoriToriVtra genes32 kb100 kbused to initiate replication for transferused to initiate plasmid replicationIS elements (insertion sequences used in

30、 transposition)F-plasmid integrated into chromosome in rare instances: two mechanisms.1-homologous recombination2- transpositionDiscrete region that has transfer genes:tra & trb loci (40 genes)(Origin of transfer)oriT traM J YALEKBPVRC WU N trbCDE traF trbB traH G ST D I/ZfinPTransfer genestraJactiv

31、atortraY/traITranscription unittra region of the F plasmidFig. 13.20Genes VIIIDirection of transferregulationtra & trb loci;40 genesExpressed coordinately as a part of single transcription unit traY/traIoriT traM J YALEKBPVRC WU N trbCDE traF trbB traH G ST D I/ZfinPTransfer genestraJactivatorpilinS

32、urface exclusionChannel?Negative regulator transcripttraY/traIDNA nicking and unwindingtra region of the F plasmidFig. 13.20Genes VIIISenses that mating pair formedDirection of transferregulationtraT-outer membrane protein that blocks mating pair formation.traS-blocks DNA transfer.traI- covalently a

33、ttaches to 5 end of DNA & unwinds itfinOHelps finPtraY- recruits traI to 5 end of DNA1. A site on the plasmid, known as the origin of transfer (oriT) is nicked by a specific endonuclease (one of the tra gene products).2. A pore is formed between the two cells and only ONE strand of DNA is passed thr

34、ough to the other cell (5 end first).53. The single strand in each cell undergoes replication to form double stranded DNA.Mechanism of self-transmissible transferOnly a single unit length of F factor is transferredF+F-F+F+Free 5 endF pili are essential for initiating pairing but are NOT channels for

35、 DNA transportDNA transferred through channel formed by protein coded by traD gene.质粒DNA的迁移作用 质粒的迁移作用(mobilization)同接合型质粒的自我转移过程是属于两种不同的概念。 非接合型的质粒由于分子小不足以编码全部转移体系所需要的基因，因而不能够自身转移。但如果在其寄主细胞中存在着一种接合型的质粒，那么它们通常也是可以被转移的。这种由共存的接合型质粒引发的非接合型质粒的转移过程叫做质粒的迁移作用。 ColE1是一种可以迁移但是属子非接合型的质粒。遗传学的研究已经揭示出，ColE1质粒从给体细

36、胞转移到受体细胞的生化过程，需要质粒自己编码的2种基因参与。 一个是位于ColE1 DNA上的特异位点 bom；另一个是ColE1质粒特有的弥散的基因产物，即 mob 基因编码的核酸酶。Mechanism of plasmid mobilization1. The mob plasmid cannot transfer without another plasmid2. The other plasmid may or may not be a self-transmissible plasmid but MUST contain tra functions (cell contact, ni

37、cking).3. If the other plasmid is self-transmissible it may also transfer.tratratratratramob mob基因和bom基因参与ColEl质粒的迁移作用这个纶论，是根据下面的实验结果作出的。相容性的2种质粒F和ColEl共存于同一细菌细胞中，F质粒可以为ColEl质粒提供它所缺乏的结合功能，这样使得ColEl质粒也能够发生转移作用。图A中表示位于F- 细胞中的ColEl质粒的状态，它的mob基因进行了转录，其产物使bom位点发生单链断裂，而出现缺口，于是，ColEl DNA便从超盘旋的结构转变成为缺口环状的构型

38、。但ColE1质粒缺乏形成性须的能力，无力进行结合配对，所以它的DNA也就不能从一个细胞转移到另一个细胞。正是由于不能够发生转移，这种从超盘旋到缺口环状的构型转变过程就有可能被回复，所以就出现这两种构型之间的平衡状态。图B中的细胞同时含有F和ColE1两种质粒。F因子能够导致性须的合成，为其DNA转移提供了转移装置，因此ColE1可以被转移。而当F质粒提供的这种转移装置被分离掉的情况下，ColE1的mob- 突变体便不能够转移。遗传分析证明mob- 突变是隐性的，mob基因编码一种蛋白质。而且当这种突变体质粒被分离出来时，并不是以松弛复合物的形式存在。图C所示 F质粒无力帮助mob- 突变体进

39、行转移，其中F性须和转移装置虽巳形成，但ColE1 DNA并没有发生缺口。图D表示另一种具mob+ 表型并带有一个顺式显性突变的ColE1突变体，它缺失了bom位点。在这样的寄主细胞中，虽然能够合成mob蛋白，但由于不能够发生缺口，因此仍然不能够转移。生物安全从ColE1质粒或其亲缘关系密切的pMBl质粒派生的绝大多 数的克隆载体，都已经丧失掉了编码迁移蛋白质的DNA区 段。但是这种蛋白质可以由亲和性质粒，例如ColK的tran 基因来补充 实验中使用的许多载体分子，在其构建过程中都已经丢掉 了nic位点，因此它们便不能够被迁移。 要使这类质粒发生接合转移的唯一可能途径是，通过重组作用形成融合

40、的或共整合的质粒，从而使它们在形体上变成接合型质粒的一部分。 应用诸如recA这样的重组缺陷的寄主菌株，便可排除这种可能性。因此，在recA 寄主中的失去nic位点的质粒载体，在生物学上要比 nic+ 质粒载体较为“稳定”。质粒DNA的复制类型 根据寄主细胞所含的拷贝数的多少可将质粒分成两种不同的复制型。 一种是低拷贝数的质粒,每个寄主细胞中仅含有13份的拷贝，我们称这类质粒为“严紧型“复制控制的质粒(Stringent plasmid)； 另一类是高拷贝数的质粒, 每个寄主细胞中可高达1060份拷贝，这类质粒被称为“松弛型”复制控制的质粒(relaxed plasmid)。 质粒的接合转移能

41、力，同它们的分子大小及复制类型之间存在有一定的相关性。一般说来，接合型的质粒具有较高的分子量, 每个细胞中仅有少数几份拷贝, 属于严紧型的；而非接合型的质粒，则往往具有较低的分子量, 每个细胞中含有较高的拷贝数, 属用于松弛型的。 一种质粒究竟是属于严紧型还是松弛型并非绝对的，这往往同寄主状况有关。pMB1 and ColE1 repliconsRNA IIRNA Irop geneRop protein (63 amino acids)Direction of DNA replication -500 -300 -100 ori 100 300 500RNAse H ProcessingTh

42、e RNA-DNA hybrid is cleaved by RNaseH to generate a free 3-OH end which serves as a primer for DNA polymerase I to initiate replication. DNA polymerase III takes over after a short distance.The RNA-DNA hybrid is NOT cleaved by RNaseH and transcription continues nonproductively. The choice between th

43、ese fates depends on the formation of a specific secondary structure in the nascent RNA II transcript. If the structure forms correctly then RNaseH processing occurs correctly; if not, it does not. Clearly, anything that interferes with the formation of the correct secondary structure will interfere

44、 with plasmid replication. There is a specific RNA whose function is to do just that! RNA I is a 108 nt molecule which is transcribed - in the opposite direction to RNA II - from a promoter located between the RNA II promoter and the origin of replication:Since RNA I is perfectly complementary to RN

45、A II, it is able to form a stable RNA-RNA hybrid with it. If it does, then RNA II will not be able to function as a primer.This is an example of antisense RNA regulation.Replication of ColE1 also depends on the Rop protein.Rop is a small protein (63 aas) which increases the rate of binding of RNA I

46、to RNA II. If Rop is absent, plasmid replication will be more frequent.pMB1 and ColE1 RepliconsSo what will happen if we alter RNA I or rop?Decrease negative regulation of RNA IIMore RNA II availableMore plasmid replicationExamplepUC plasmids have a single mutation (G-A) one nucleotide upstream of t

47、he initiation of RNA I.pUC plasmids have 500-700 copies per cell The relative copy numbers of pBR322 and pUC18 illustrate the role of Rop perfectly. There are approximately 15 copies of pBR322 per cell; however, there are 50-100 copies of pUC18, which is derived from pBR322. The relevant difference

48、between the two plasmids is the fact that pBR322 contains the rop gene but pUC18 does not.质粒的亲和性Plasmid compatibility the ability of two different plasmids to co-exist in the same hostPlasmids that utilize the same replication system cannot co-exist in the same bacterial cellPlasmids carrying the sa

49、me replicon belong to the same incompatibility group2. 大肠杆菌质粒载体 pSC101质粒载体 9.09kb 严紧型复制控制的低拷贝数质粒。 pSCl01质粒是从接合型质粒R6-5派生出来的，而R6-5又是分子大小为97kb的R6质粒的一种衍生物。 Stanley N. Cohen 1973获得的。 R6-5 DNA经流体切割环化，转化大肠杆菌，四环素抗性转化子中分离出来的. R6质粒携带有抗链霉素、磺胺、氯霉素、卡那霉素及四环素等多种抗性基因。在R6-5质粒上四环素抗性基因已经丧失，但仍保留者其它几种药物抗性基因。 克隆 非洲爪蟾 ( X

50、enopus leavis ) rDNAColE1质载体 松弛型复制控制的多拷贝的质粒 编码有大肠杆菌素E1基因(cea) 还编码有使寄主细胞具有对大肠杆菌素E1免疫性的基因 imm E1 在EcoRI位点上插入外源DNA，虽然使它失去了产生大肠 杆菌素E1的能力，但却不影响其DNA复制活性，以及对 大肠杆菌素E1的免疫性能。对大肠杆菌素的免疫性特征可作为一种选择标记。以大肠杆菌素E1为例，这种蛋白质是由Co1E1质粒控制合成的，它对于不含有Co1E1质粒的敏感细胞有致死效应，而对于带有Co1E1质粒的细胞则无此反应。我们可以应用类似于检测噬菌体的方法，来检测大肠杆菌素EI的产生：将产生大肠杆

51、菌素E1的寄主细胞涂布在由敏感细菌生长的菌苔上，由寄主细胞分泌出来的大肠杆菌素E1，会抑制周围敏感细胞的生长并使之致死，于是便会在看起来显得混浊不透明的菌苔背景上出现空班的清亮区。 但如果在Co1E1质粒的EcoRI位点上插入一段外源DNA，由于这个位点恰好是位于大肠杆菌素E1基因的编码序列内部，因此，这种插入作用导致了该基因的失活。携有这种重组质粒的寄主细胞就不能够合成大肠杆菌素E1( Co1E1- )，但它的大肠杆菌素E1免疫基因仍有活性，照样表现出E1免疫性的表型(Imm E1+ )。所以Co1E1质粒编码的大肠杆菌免疫基因Imm E1，可以作为此质粒的选择记号，而Co1E1- 的表型则

52、为在 EcoRI 位点带有插入序列的Co1E1重组质粒，提供了有效的选择标记。pBR322 Bolivar and RodriguezBolivar et al.(1977a,b) The molecular origins of plasmid pBR322.R7268 was isolated in London in 1963 and later renamed R1.1, A variant, R1drd19, which was derepressed for mating transfer, was isolated. 2, The ApR transposon, Tn3, fro

53、m this plasmid was transposed on to pMB1 to form pMB3. 3, This plasmid was reduced in size by EcoRI* rearrangement to form a tiny plasmid, pMB8, which carries only colicin immunity. 4, EcoRI* fragments from pSC101 were combined with pMB8 opened at its unique EcoRI site and the resulting chimeric mol

54、ecule rearranged by EcoRI* activity to generate pMB9. 5, In a separate event, the Tn3 of R1drd19 was transposed to Col E1 to formpSF2124. 6, The Tn3 element was then transposed to pMB9 to form pBR312. 7, EcoRI* rearrangement of pBR312 led to the formation of pBR313, from which (8) two separate fragm

55、ents were isolated and ligated together to form pBR322. During this series of constructions, R1 and Col E1 served only as carries for Tn3. (Reproduced by courtesy of Dr G. Sutcliffe and Cold Spring Harbor Laboratory.)pSF2124 R1drd19 与 ColE1 共培养 Tn3 从 R1drd19 转座到ColE1形成。Tn3携带ApR -内酰胺酶基因。R1drd19 R1变异体

56、 携带：AmpR、CmlR、StrR、SulR、KanR。 R1 即 R7268 1963 在英国伦敦 从沙门氏菌分离得到，携带 AmpR ，后改名为R1 。pMB1 8.3kb pMB3 13.3kb Tn3 从 R1drd19 转座到pMB1 EcoRI* -AATT- ligate and rescuepMB8 2.6kb，带大肠杆菌素EI免疫性基因及EcoRI单一识别 位点，但失去了ApR。pMB9 5.3kb (pSC101 EcoRI* ) + ( pMB8 EcoRI ) ligatepBR312 10.2kb, Tn3 从 pSF2124 易位到 pMB9。 pBR313 8.

57、8kb pBR312 EcoRI* ligate 消去一个BamHI。 pBR318 pBR313消去两个PstIpBR320 pBR313 去 EcoR II 片段pBR322 4363bp pBR318 pBR320 酶切重组。 Cloning vector pBR322, complete sequence ORIGIN 1 ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa 61 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcg

58、g 121 caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt 181 gcgggatatc gtccattccg acagcatcgc cagtcactat ggcgtgctgc tagcgctata 241 tgcgttgatg caatttctat gcgcacccgt tctcggagca ctgtccgacc gctttggccg 301 ccgcccagtc ctgctcgctt cgctacttgg agccactatc gactacgcga tcatggcgac 361 cacacccgtc ctg

59、tggatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac 421 aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca 481 cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg 541 actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct 601 caacctacta ctgggctgct tcctaatgca g

60、gagtcgcat aagggagagc gtcgaccgat 661 gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt 721 cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct 781 ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct 841 tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc