1. Bacterial and Viral Genetics 어제 … DNA replication DNA repair genetic rearrangement leading strand proofreading DNA recombination lagging strand repair system transposon 어제 … DNA replication DNA repair genetic rearrangement leading strand proofreading DNA recombination lagging strand repair system transposon Transformation : 형질 전환 Transduction : 형질 도입 Replica plating : 복제 평판법

2. Gene Transfer and Genetic Recombination in Bacteria  Genetic recombination occurs in E. coli ( 자주 )  Bacterial conjunction brings DNA of two cells together, allowing recombination to occur  Transformation ( 형질전환 ) and transduction( 형질도입 ) provide additional resources of DNA for recombination  Replica plating ( 복제평판법 ) allows genetic recombinants to be identified and counted

3. Genetic Recombination in Bacteria  Some bacteria genetically reshuffle Genes transferred from one individual to another recombine with existing DNA  Bacteria, including E. Coli, can be grown on minimal media (water, organic C, salts with N) Minimal media provide laboratory ease Genetically identical clones allow molecular genetics studies

4. Notation of Recombination ( 극한 조건 ) Vitamin biotin amino acid methionine Amino acids leucine threonine, vitamin thiamine Three letter gene name, + normal, – mutated allele Several hundred out of 100 million grew

5. Colonies: bio + met + leu + thr + thi + Minimal medium Mutant strain 1: bio – met – leu + thr + thi + Mixture of strains 1 and 2 Mutant strain 2: bio + met + leu – thr – thi – No colonies

6. Conjugating E. coli Cells Recipient cell lacking F factor Recipient cell lacking F factor Donor cell with F factor Donor cell with F factor Sex pilus Sex pilus

7. Bacterial Conjugation  Bacterial recombination occurs by conjugation Sex pilus ( 성선모 ) connects two bacteria Donor sends DNA via cytoplasmic bridge to recipient  Recipient undergoes recombination Plasmids: Circular, nonchromosomal transferable DNA R plasmids confer resistance to antibiotics

8. Plasmid Disrupted Bacterial Cell

9. F Factor and Conjugation  Donor cell must have F factor (fertility) plasmid F + cells are donors with F factor F - cells are recipients without F factor  F factor has genes that encode for sex pilus Sex pilus cytoplasmically connects F + to F - F - coverts to F + after receiving and synthesizing new DNA, no recombination occurs

10. Bacterial chromosome Transfer of the F factor Transfer of the F factor F factor 1 F+F+ F–F– One strand of the F factor breaks at a specific point and begins to move from F + (donor) to F – (recipient) cell as the F factor replicates. DNA replication of the F factor continues in the donor cell, and a complementary strand to the strand entering the recipient cell begin to be synthesized. When transfer of the F factor is complete, replication has produced a copy of the F factor in both the donor and recipient cells; the recipient has become an F +. No chromosomal DNA is transferred in this mating. 2 3 4 An F + cell conjugates with an F – cell.

11. Hfr Cells and Recombination  Hfr cells integrate F factor into bacterial chromosome through recombination Hfr cells can conjugate with F - cells Recipient becomes partial diploid  Genetic recombination occurs by double crossing over in recipient New generations have recombined DNA

12. Transfer of Bacterial Genes

13. Mapping Genes by Recombination  Full DNA transfer by conjugation takes 90 to 100 minutes Partial DNA transfer when sex pilus breaks Timing of DNA transfer allows mapping of E. coli chromosome, map units are minutes  Order and timing of DNA transfer show E. Coli has circular chromosome Direct sequencing confirmed genetic mapping

14. Transformation ( 형질 전환 )  Transformation occurs when bacteria take up DNA from disintegrated bacteria Linear fragments recombine by double crossovers Transformation bacteria usually have DNA binding protein in wall  Genetic manipulation of some bacteria requires artificial transformation Alters cell membrane for DNA pentration Electroporation

15. Transduction ( 형질도입 )  Transduction occurs when bacterial phages transfer DNA from one bacteria to another  Virus incorporates DNA fragments from host cell If DNA fragments are homologous, bacteria become partial diploids Recombination by double crossovers

16. Replica Plating ( 복제 평판법 )

17. Replica Plating  Replica plating identifies and counts genetic recombinations in bacterial colonies Master plate pressed onto sterile velveteen Velveteen pressed onto replica plates with different growth media  Complete medium has full complement of nutrient substances Auxotrophic mutants will not grow on media missing particular nutrients

18. Viruses and Viral Recombination  Viruses in the free form consist of a nucleic acid core surrounded by a protein coat  E. coli’s bacteriophages are widely used in genetic research

19. Viruses in Free Form  Free form viruses are outside host cell Nucleic acid core Protective protein coat Some animal viruses have envelope from cell membrane  Viruses can not do own metabolism Subvert host cellular machinery for DNA replication and protein synthesis

20. Viruses

21. E. coli’s Bacteriophages  Virulent bacteriophages kill host bacteria during each cycle of infection Temperate bacteriophages have inactive phase of DNA replication with host cells  T-even bacteriophages (T2, T4, T6) are most valuable in E. coli studies Head has linear molecule of DNA Tail attaches to E. coli

22. T-Even Infective Cycle

23. Viral Lytic Cycle  Lytic cycle Series of events from viral infection to release of viral progeny  Generalized transduction Occurs from random incorporation of host DNA fragments

24. E. coli Lambda Bacteriophage  Lamba (λ) E. coli bacteriophage Typical temperate phage with two paths Lytic cycle goes directly from infection to progeny virus release  Lysogenic cycle integrates λ chromosome into host Insertion at specific sequences, then crosses over Prophage viral genome inactive until trigger Specialized transduction transfer of host genes near λ genome

25. Lambda Infective Cycle

26. Transposable Elements (TE: 전위인자 )  Insertion sequence elements and transposons major types of bacterial transposable elements  Transposable elements were first discovered in eukaryotes  Eukaryotic transposable elements are classified as transposons or retrotransposons  Retroviruses are similar to retrotransposons

27. Transposable Elements  Transposable elements (TEs) Segments of DNA that move around cell genome Transposition is movement of TEs, jumping gene Target site of TE is not homologous with TE No crossing over  TEs can move in two ways Cut-and-paste, original TE leaves Copy-and-paste, original TE stays in place

28. Cut-and-Paste Transposition

29. Copy-and-Paste Transposition

30. Bacterial Transposable Elements

31.  Insertion sequences Contain only genes for tranposition Transposase catalyzes insertion or removal Inverted repeats mark insertion sequence (palindromic repeat)  Transposon Multiple genes within inverted repeat sequences Antibiotic resistance genes within transposons Transposons can move to plasmids

32. Transposons  Eukaryotes have no TEs resembling insertion sequences  Eukaryotic transposons are similar in structure and function to prokarytotic Use transposase and inverted repeat sequences Use cut-and-paste or copy-and-paste

33. Retrotransposons  Retrotransposons transpose by copy-and-paste but transposition occurs via intermediate RNA Retrotransposon transcribed into RNA Reverse transcriptase uses RNA to make DNA DNA copy inserted into DNA at new location  TE-instigated genes occur within a TE and lose normal expression controls Linked to certain forms of cancer

34. Retrotransposon

35. Retroviruses  RNA genome replicates with DNA intermediate Uses reverse transcriptase  Common in vertebrates Retrovirus genomes common in humans HIV and carcinogenic retroviruses

36. Retrovirus first or retrotransposon first?  아마도 retrotransposon 이 먼저였을 것이다. 이것이 virus 화함.  왜냐하면, alu 라고 하는 retrotransposon 은 진핵세포에서 발견, virus 에서는 발견안됨.  이러한 retrotransposon 이 많음  인간 유전체의 40% 차지 (cut & paste)