Molecular Biology Xu Liyan Chapter 14 gene recombination and gene engineering

section 1 DNA Recombination section 2 Recombinant DNA technology section 3 Relationship between Recombinant DNA technology and Medicine

section 1 DNA Recombination 1.1 Homologous Recombination 1.2 Gene Transfer and Recombine in Bacteria Conjugation Transformation Transduction 1.3 Site-specific Recombination 1.4 Transpositional Recombination

1.1 Homologous Recombination The covalence connection between different DNA moleculars is called DNA recombination or gene recombination The gene recombination includes two types as follows  homologous recombination  site-specific recombination  transpositional recombination

 Homologous Recombination The recombination between homologous sequence is known as homologous recombination

Mechanism of Homologous Recombination 5’ 3’ Rec BCD Rec A Ligase Holliday Mechanism of Homologous Recombination

5’ 3’ Ruv C Ligase Holliday

1.2 Gene Transfer and Recombine in Bacteria Conjugation 3 4 5 n F- F+ 1.2 Gene Transfer and Recombine in Bacteria Conjugation F factor

Transformation  There is foreign DNA.  The phenotype of organisms is changed.  The changed Phenotype is passed down stably

transformation of diplococcus pneunonine DNA Diplococcus pneumoniae with capsule is toxicity to human Diplococcus pneumoniae without capsule is untoxicity to human. transformation of diplococcus pneunonine A well-known experiment Avery, 1943

Transduction one cell and go to infect other cell, the  When virus is released from infected one cell and go to infect other cell, the DNA fragment transfer from one cell to other cell. This is called the transduction.

bacteria 1 integration bacteria 2 phage integration some factor bacteria 2

 DNA fragment transformation between two bacterium  phage is carrier bacteria 1 phage integration  DNA fragment transformation between two bacterium  phage is carrier some factor bacteria 2

two site-specific DNA fragments is known as 1.3 site-specific recombination The integration catalyzed by integrase between two site-specific DNA fragments is known as site-specific recombination.

1.4 Transpositional recombination the displacement of some gene in the genome by insertion sequence or transposons

1.4.1 insertion sequence and its mediated gene transposition  The length of typical insertion sequence is about 750~1500bp.  Typical insertion sequence includes two 9 ~ 41bp inverted repeat sequence and a transposase.  A 4 ~12bp positive repeat sequence always link to flanking of inverted repeat sequence.  Gene transposition by insertion sequence:  conservative transposition  duplication transposition

duplication transposition target transposase insertion sequences 3’ 5’ polymerase I ligase duplication transposition 1 2

1.4.2 structure of transposons  The transposon is a dispersive and repeat sequence in the genome.  The transposon can transfer from one region to other region of the genome.

transposase gene and flanking inverted repeat  The structure of transposon is similar to one of insertion sequence.  The both insertion sequence and transposon contain transposase gene and flanking inverted repeat sequences, but transposon also contain a few other genes.  The insertion sequence is the most simple transposon in fact.

insertion sequence Tn3 Tn10 IS 10L amp-R gene repressor gene transposase insertion sequence transposase gene Tn3 tet-R gene Tn10

section 2 DNA recombination technology 2.1 the basic concept related with DNA recombination technology 2.2 the basic principle of DNA

recombination technology 2.1 the concept related with DNA recombination technology 2.1.1 DNA cloning 2.1.2 tool enzyme 2.1.3 target gene 2.1.4 gene vector

2.1.1 DNA cloning  It is a process of DNA molecular amplification.  Usually, the first a target DNA fragment is inserted to a vector and a recombinant (replicon) is constructed.  The second the recombinant is transformed into host cell and screen out the cell containing the recombinant.  The last that cell is amplified, namely a mass of target DNA molecule is gained.

2.1.2 tool enzyme  restriction endonuclease  DNA ligase  DNA polymerase I  reverse transcriptase  polynucleotide kinase  end-transferase  alkaline phosphatase

structural character of cutting site recognized by restriction enzyme restriction endonuclease recognized sequence and cut Bam H I Eco R I GAATTC CTTAAG GGATCC CCTAGG 5’ Pvu I Sst I GAGCTC CTCGAG CGATCG GCTAGC Alu I Sma I AGCT TCGA CCCGGG GGGCCC

2.1.3 target gene The interested gene is the target gene

source of the target gene * It is from genomic DNA directly, this is prokaryotic gene only generally. * It is from artificial synthesis, this is simple polypeptide gene generally. * It is from mRNA. * It is from genomic library or cDNA library. * Polymerase Chain Reaction (PCR).

? synthesize cDNA from mRNA S1 nuclease mRNA DNA polymerase I reverse AAA…AAA 5’ 3’ mRNA TTT...TTT primer: oligo dT reverse transcriptase basic hydrolysis ? DNA polymerase I S1 nuclease

genomic library cDNA library genomic DNA fragment 50-200kb extraction restrictively cut gene fragments recombination recombinant transformation cDNA library target gene extraction mRNA cDNA double strands recombination recombinant transformation cDNA library

3' 5' PCR Process denaturation annealing Next cycle extension 5' 3'

2.1.4 gene vector which structure is reconstructed.  The gene vectors are DNA molecules, which structure is reconstructed.  They can carry target DNA fragment  The target gene or DNA fragment is amplified and expressed.

* herpes simplex virus DNA * Epstein-Barr virus DNA vector * plasmid * cosmid *  phage * M13 phage * insect virus DNA (autograph californica virus , ACNPV) * yeast artificial chromosome DNA * vaccinia virus DNA * simian virus 40 DNA 3-10kb 40kb 29-48.5kb 5.243kb 180kb 6.407kb 20kb 4-8kb 15kb 0.3-1.0kb 2.5kb 128kb 100kb 25kb * bovine papilloma virus DNA 8.0kb 10kb 0.2-2.2Mb 0.3-1.2Mb * retrovirus DNA * fowlpox virus DNA * adenovirus DNA * herpes simplex virus DNA * cytomegalovirus DNA * Epstein-Barr virus DNA 240kb 170kb 6.407kb 233-238kb 8-10kb 24-36kb

A single restriction site Xmn I 3966 2034 Xmn I Pst I 3612 2067 Pvu II 1424 Ava I 650 Sal I 375 BamH I plasmid pBR322 4.36kb 29 Hind III EcoR I 0 A origin A screening gene A single restriction site condition tetr ampr ori

plasmid pUC19 Plac 2.69kb ori lac Z’ polylinker 52bp ampr lac I Eco R I Sac I Kpn I Sma I Bam H I Xba I Hinc II Pst I Sph I Hind III ampr ori polylinker 52bp Plac lac I lac Z’

2.2 the basic principle of DNA recombination technology

the procedure of gene cloning separate target gene as well as vector 1 cut target gene and vector restrictedly 2 join target 3 recombinant transformation 4 ligate target screening 5 go a step further... target gene amplify 6

separate target gene incomplete digestion Sma I complete incomplete 1 2 3 1+3 incomplete digestion Sma I complete 1+2+3 1 ------ 2, 2 2 ------ 5, 3 3 ------ 9, 4 4 ------ 14, 5 5 6 n n+n(n+1)/2 n+1

cut and ligate target gene and vector CCGG GGCC plasmid Hpa II CCGG GGCC Hpa II genome DNA CGG C GGC Hpa II CGG C GGC Hpa II CCGG GGCC Ligase recombinant

vectors and recombinants recombinant transformation vectors and recombinants competent cells

recombinant screening amp or tet etc + plasmid extraction digist with restriction enzeme 1 2 marker -

target gene amplification

食管癌细胞NGAL基因5’端转录调控区不同长度片段PCR扩增结果 200bp M 1431 1137 945 657 416 152 1124 2000bp  1000bp 

重组子pGEM-1431~152 XhoI和BglII双酶切后，琼脂糖凝胶电泳鉴定结果 200bp M1 1431 1137 945 657 416 152 1124 M2 1000bp  ← 947 bp ← 5.0 kb ← 2.0 kb

重组子pGLP-1431~152 XhoI和BglII双酶切后，琼脂糖凝胶电泳鉴定结果 M 1431 1137 945 657 416 152 5000bp  1375 bp  564bp 

target gene expression prokaryotic expression system D

Expression analysis of four expression vectors in E.coli by SDS-PAGE

eukaryotic expression system 21kDa → ← 25kDa 1 2 3 4 5 6 7 8 9 10

section 3 the relationship between DNA recombination technology and medicine  discover and separate pathogenic gene  biopharmacy  DNA diagnosis  gene therapy  prevent transmissibility disease

Summary Transposition DNA cloning: separate , cut, ligate , Homologous Recombination Site-specific Recombination Transposition Conjugation Transformation Transduction DNA cloning: separate , cut, ligate , transform, screen, amplify, express

选择题练习 基因重组与基因工程

1. 基因工程的特点是 A 在分子水平上操作,在分子水平上表达 B 在分子水平上操作,在细胞水平上表达 C 在细胞水平上操作,在分子水平上表达 D 在细胞水平上操作,在细胞水平上表达 E 以上均可以

2. 限制性核酸内切酶不具有哪项特点 ? A 仅存在于原核细胞中 B 用于重组DNA技术中的位I类酶 C 能识别双链DNA中特定的碱基顺序 D 具有一定的外切酶活性 E 辨认得核苷酸序列常具有回文结构

3. 有关质粒的叙述,下列哪项是错误的 ? A 小型环状双链DNA分子 B 可小到2-3kb, 大到数百个kb C 能在宿主细胞中独立自主地进行复制 D 常含有耐药基因 E 只有一种限制性核酸内切酶切口

4. 下列哪项不是重组DNA的连接方式? A 粘性末端与粘性末端的连接 B 平端与平端的连接 C 粘性末端与平端的连接 D 人工接头连接 E 同聚物加尾连接

5. DNA克隆不包括下列哪项步骤? 选择一个适合的载体 限制性核酸内切酶在特异位点裂解质粒和目的基因 用连接酶连接载体DNA与目的DNA,形成重组体 用载体的相应抗生素抗性筛选含重组体的细菌 重组体用融合法导入细胞

6. 下列哪种酶是重组DNA技术中最重要的? A 反转录酶 B 碱性磷酸酶 C 末端转移酶 D DNA聚合酶I E DNA连接酶

7. 基因工程中通常使用的质粒存在于 A 细菌染色体 B 酵母染色体 C 细菌染色体外 D 酵母染色体外 E 以上均不是

8. 在已知DNA序列情况下,获取目的DNA最方便的方法是 B 基因组文库法 C cDNA文库法 D PCR法 E 从染色体DNA直接提取

9. 基因工程中使目的基因与载体拼接的酶是 A DNA聚合酶 B RNA聚合酶 C DNA连接酶 D RNA连接酶 E 限制性核酸内切酶

10. 表达人类蛋白质的最理想的细胞体系是 A E.coli 表达体系 B 原核表达体系 C 酵母表达体系 D 昆虫表达体系 E 哺乳类细胞表达体系

11. The nucleotide number which restriction enzyme recognize in DNA nucleotide sequence is A 4, 5 or 6 B 5, 6 or 7 C 6, 7 or 8 D 4, 6 or 8 E 4 - 8

12. The technique used in identification of DNA is A northern blotting B southern blotting C Western blotting D affinity chromatography E ion exchange chromatography

13. The way of gene recombination doesn’t include A transformation B transduction C transposition D change-over转换 E integration

14. The abbreviation of polymerase chain reaction is A PRC B PER C PDR D BCR E PCR

15. 对于重组体的筛选,属于非直接选择法的是 A 免疫化学法 B 原位杂交法 C southern 印迹 D 补救标志筛选 E 酶联免疫筛选

16. 基因工程中,目的基因地来源有 A 化学合成 B PCR合成 C cDNA文库 D 基因组文库 E 组织细胞中染色体DNA直接提取

17. 质粒DNA等作为基因工程载体必须具备的条件是 B 易转化 C 易筛选(质粒DNA含有抗药性基因等) D 具有合适的限制性核酸内切酶酶切位点 E 易提取获得

18. 将表达载体导入真核细胞的转染方法有 A 磷酸钙转染 B DEAE葡萄糖介导转染 C 电穿孔 D 脂质体转染 E 显微注射

19. gene cloning also be called A DNA recombination B RNA recombination C DNA cloning D RNA cloning E protein replication

20. The enzyme tools commonly used in gene cloning technique are A restriction enzyme B DNA polymerase I C DNA ligase D reverse transcriptase E terminal transferase