Lecture 27

1. Lecture 27 第10章 重组 10.1 同源重组 10.1.1 交换机理 10.1.2 双链断裂 修复机理 10.1.3 RecBCD途径 10.1.4 基因转换 10.2 非同源重组 10.2.1 转座子 10.2.2 反转录转座子 10.2.3 λ噬菌体的整合 Chapter 10 Recombination 10.1 Homologous Recombination 10.1.1 Mechanism for Crossing-Over 10.1.2 Mechanism for Double- Stranded Break Repair 10.1.3 The RecBCD pathway 10.1.4 Gene Conversion 10.2 Non-homologous Recombination 10.2.1 Transposons 10.2.2 Retrotransposons 10.2.3 Bacteriophage λ integration

2. Review of Lecture 26 A1→Homologous Recombination 同源重组 A2-1→ Resolution of the Holliday junction to create a crossover产生交换的Holliday交叉拆分 A2-2→ Resolution of Holliday junctions for non-crossover不产生交换的Holliday交叉拆分 A3→ The RecBCD Pathway / RecBCD途径

3. A1→Homologous Recombination 同源重组 同源重组：在具有相似DNA序列的DNA分子之间发生的重组。 Homologous recombination:Recombination between DNA molecules with similar DNA sequences.

4. A2-1→ Resolution of the Holliday junctionto create a crossover产生交换的Holliday交叉拆分

5. A2-2→ Resolution of Holliday junctionsfor non-crossover不产生交换的Holliday交叉拆分

6. A3→ The RecBCD Pathway RecBCD途径 RecBCD途径：大肠杆菌中一种由RecBCD蛋白启动的主要的同源重组途径。 RecBCD pathway:The major homologous recombination pathway in E. coli, initiated by the RecBCD protein. RecBCD蛋白：大肠杆菌中能在DNA的χ位点产生切口的蛋白质。 RecBCD protein:The protein that nicks one of the DNA strands at a chi site to initiate homologous recombination in E. coli.

7. Questions for Lecture 27 Q1→ What is gene conversion? 什么是基因转换？ Q2→ What are transposons? 什么是转座子？ Q3→ What are retrotransposons?什么是反转录转座子？

8. Vocabulary of Lecture 27 (1/1) site-specific recombination attachment site integrase excisionase integration host factor, IHF transposase resolvase replicative transposition conservative transposition staggered ends direct repeat retrotransposon 位点特异性重组 附着位点 整合酶 切除酶 整合宿主因子 转座酶 解离酶 复制型转座 保守型转座 交错末端 同向重复（序列） 反转录转座子

9. Q1→ What is gene conversion?什么是基因转换？

10. A1→ 10.1.4 Gene Conversion / 基因转换 基因转换：同源重组的后果，在不同等位基因之间的杂交使其中的一个等位基因被转换成了另一个等位基因。这是由错配修复引起的。 Gene conversion:Consequence of homologous recombination, in which hybridization between different alleles causes one allele to be converted into the other. This occurs by mismatch repair. 等位基因：同一基因的不同版本，它们之间在功能和序列上稍有不同。 Alleles:Versions of the same gene that differ slightly in function and sequence.

11. Homologous, not identical同源的，而不是相同的 Homologous, not identical. G C T A 3’ 5’ 5’ 3’ G T T A 5’ 3’ 3’ 5’ Chapter 10, page 225

12. Gene Conversion / 基因转换 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’

13. Neurospora crassa 粗糙脉孢菌 + + 4 : 4 + + + + + + + + m m m m m m m m m Meiosis I 减数分裂I Meiosis II 减数分裂II m Mitosis 有丝分裂

14. Neurospora crassa 粗糙脉孢菌 4 : 4 6 : 2 5 : 3 www.neurosporagenome.org/ www.nigms.nih.gov/Initiatives/Models/ www.neurosporagenome.org/

15. 10.2 Non-homologous Recombination非同源重组 Non-homologous recombination:Rearrangement of DNA regions that are not similar (homologous) to each other. 非同源重组：互相不相似的（即不同源的）DNA区域之间的重排。 10.2.1 Transposons 转座子 10.2.2 Retrotransposons反转录转座子 10.2.3 Bacteriophage λ integration λ噬菌体的整合

16. Q2→ What are transposons?什么是转座子？

17. A2→ 10.2.1 Transposons / 转座子 转座子：基因组中能改变位置或位点的DNA元件。 Transposons:DNA elements that can change positions, or locations, in the genome. 插入序列：简单的原核生物转座子。它们是一些能使自身插入到一段DNA随机位置中的序列。 Insertion sequences:Simple prokaryotic transposons that insert themselves into random sites on a piece of DNA.

18. Complex transposons / 复杂转座子 Inverted repeat orientation: e.g. Tn10 23 722 23 23 722 23 Antibiotic resistance IS1 IS1 Direct repeat orientation: e.g. Tn9 23 722 23 23 722 23 Antibiotic resistance IS1 IS1

19. More complex transposons / 更复杂的转座子 tnpA tnpRbla res IR IR Transposase 转座酶 Resolvase 解离酶 β-lactamase β内酰胺酶 (Ampr) Organization of the Tn3 transposon 转座子Tn3的组成

20. Transposition / 转座 What is the other name(s) for transposon?

21. Simplified mechanism of transposition简化的转座机理

22. Direct repeats / 同向重复序列 同向重复序列：完全相同并以相同方向出现的序列。 Direct repeats:Sequences that are exactly the same, and present in the same orientation. 反向重复序列：互补序列从反方向读与它自身相同的序列。 Inverted repeats:Sequences that are the same if you take the complement of one and read it backwards.

23. Replicative transposition / 复制型转座

24. Conservativetransposition / 保守型转座

25. Q3→ What are retrotransposons?什么是反转录转座子？

26. A3→10.2.2 Retrotransposon反转录转座子 反转录转座子：一种以类似于反转录病毒机理进行转座的DNA 元件。 Retrotransposon:A transposable DNA element that transposes via a retrovirus-like mechanism. 反转录病毒：一种RNA病毒，其复制依赖于通过反转录形成的原病毒。 Retrovirus:An RNA virus whose replication depends on formation of a provirus by reverse transcription. LTR-Retrotransposons LTR反转录转座子 Non-LTR retrotransposons 非LTR反转录转座子

27. LTR-Retrotransposons / LTR反转录转座子 长末端重复序列：在反转录病毒的原病毒两端发现的由几百个碱基对组成的DNA区域。 Long terminal repeats (LTRs) :Regions of several hundred base pairs of DNA found at both ends of the provirus of a retrovirus.

28. Non-LTR retrotransposons非LTR反转录转座子 Weaver’s Molecular Biology, 3rd Edition, Figure 23.31

29. 10.2.3 Bacteriophage λ integrationλ噬菌体的整合 Lysogeny Lysis

30. Phage integration and excision attP: attachment sites of phage attB: attachment sites of bacterium

31. Now showingGene Conversion File: biophoto7 \ Introduction to Genetic Analysis \ Genetic Consequences of Meselson-Radding Model

32. Gene Conversion (1/5) In this cross of + X m, the m site corresponds to a mutated nucleotide pair. If, by chance, m becomes part of a heteroduplex, the Meselson-Radding model predicts aberrant ratios of the + : m alleles.

33. Gene Conversion (2/5) In most cases, m will not be part of a heteroduplex, so two of the four meiotic products will carry the mutation. After one round of mitosis, the resulting octad （八联体）has a 4:4 ratio of + : m.

34. Gene Conversion (3/5) At low frequency, an asymmetric heteroduplex including the m site can be generated by strand invasion, as postulated by the Meselson-Radding model. A crossover event without branch migration generates one +/m heteroduplex.

35. Gene Conversion (4/5) If the +/m mismatched base pair is not repaired, a 5:3 octad ratio is produced.

36. Gene Conversion (5/5) If the m strand of the mismatched DNA is repaired to +, a 6:2 octad ratio results.

37. Now showing again

38. Now showingReplicative transposition File: biophoto7 \Introduction to Genetic Analysis \ Replicative transposition. (5’)

39. Replicative transposition (1/9) Transposition is the movement of genetic elements, called transposons, between plasmids or genomes.

40. Replicative transposition (2/9) Shown here is a mechanism of replicative trans-position by the E. coli Tn3 transposon. A copy of Tn3 will be generated in the target plasmid.

41. Replicative transposition (3/9) The transposon is flanked by inverted repeat sequences and consists of several genes. One of these genes encodes the transposase enzyme.

42. Replicative transposition (4/9) In the target plasmid, transposase acts at the region of insertion.

43. Replicative transposition (5/9) The target plasmid is cleaved by transposase to yield staggered ends nine nucleotides apart.

44. Replicative transposition (6/9) Transposase also cleaves the plasmid containing the transposon at the ends of the transposable element. These ends are joined with the ends of the target plasmid.

45. Replicative transposition (7/9) The single-stranded regions of DNA are replicated.

46. Replicative transposition (8/9) The resulting plasmid is called a cointegrate（共整合体）. It contains the original transposon and a new copy.

47. Replicative transposition (9/9) The cointegrate is resolved into the original plasmid and the target plasmid by an enzyme called resolvase. The transposon contains a sequence called the internal resolution site, or IRS, which is necessary for resolution.

48. Now showing again

49. Essentials of Lecture 27 / 第27讲要点 A1→ Gene Conversion / 基因转换 A2→ Transposons / 转座子 A3→ Retrotransposon / 反转录转座子

50. A1→ 10.1.4 Gene Conversion / 基因转换 基因转换：同源重组的后果，在不同等位基因之间的杂交使其中的一个等位基因被转换成了另一个等位基因。这是由错配修复引起的。 Gene conversion:Consequence of homologous recombination, in which hybridization between different alleles causes one allele to be converted into the other. This occurs by mismatch repair. 等位基因：同一基因的不同版本，它们之间在功能和序列上稍有不同。 Alleles:Versions of the same gene that differ slightly in function and sequence.