1. Microbial Genetics 1 楊倍昌

2. Start with “what-and-how” questions (PR Murray, Medical Microbiology. 2013; 7 pages) 1. Mutation, Repair, and Recombination. 2. Gene Exchanges in prokaryotic cells. 3. Mechanisms of Genetic transfer between cells. 4. Recombination. 5. Genetic Engineering. Traditional outline (R Bauman, Microbiology. 2007; 64 pages) 1. The structure and replication of genomes. 2. Gene function. 3. Mutation of genes. 4. Genetic recombination and transfer. 5. Recombinant DNA technology 6. Ethics and safety Learning objective 很奇怪的排法 2

3. There is nothing different between the basic structure of substances in livings carrying heritage E. coli DNA spread Chromosome of an insect George B. Kauffman, DNA Structure: Happy 50th Birthday! http://chemeducator.org/sbibs/s0008003/spapers/830219gk.htm 3

4. 1866, Mendel published his lecture, a work that was to establish him as “the father of genetics”. 1869 Johann Friedrich Miescher (nuclein) 1873 Anton Schneider (meiosis) 1879 Walther Flemming (chromaton, mitosis) 1888 Wilhelm von Waldeyer-Hartz, (term chromosome) 1902 Walter Stanborough Sutton. (chromosomes carry the units of inheritance) 1904 Theodor Boveri (correlation between Mendel's factors and chromosomes ) 1904 William Bateson (genetics) 1909 Wilhelm Johannasen (gene) 4

5. Milestones in DNA History 1869 Johann Friedrich Miescher identifies a weakly acidic substance of unknown function in the nuclei of human white blood cells. This substance will later be called deoxyribonucleic acid, or DNA. 1924 Microscope studies using stains for DNA and protein show that both substances are present in chromosomes. 1928 Franklin Griffith, a British medical officer, discovers that genetic information can be transferred from heat-killed bacteria cells to live ones. This phenomenon, called transformation, provides the first evidence that the genetic material is a heat-stable chemical. 1944 Oswald Avery, Maclyn McCarty, and Colin MacLeod, identify Griffith's transforming agent as DNA. 1949 Erwin Chargaff reports that DNA composition is species- specific. Chargaff also finds that the amount of adenine equals the amount of thymine, and the amount of guanine equals the amount of cytosine in DNA from every species (the magic number). 1953 James Watson and Francis Crick discover the molecular structure of DNA. 化學 生物學 生化學 分子生物學 ( 物理 ) 5 略

6. Then, the day after 1953, the mystery of life becomes two coiled strands Rosalind Franklin (1920-1958) 6 略

7. Replication by “conventional manner” 7

8. Time DNA replication starts from the origin: 1. E. coli temperature sensitive in initiation were synchronized by being transferred to 40 o C for one hour, then shifted to 28 o C. 2.At different times, the culture was pulse labeled with 3 H-thymidine for 1 min 歷史問題 : On the structure and replication ( 位置 ). 8 略

9. How to make sure DNA copies have been equally distributed into daughter cells? minicells 歷史問題 : On the structure and replication ( 如何分配 ). 9 略

10. Evidence of response to environment heat shock responses of E. coli Protein synthesis:  Total proteins Stimulated proteins ○ Other proteins Gene function ( 生物化學的測量方法 ). 10

11. Control, responses, adaptation When the temperature gets too worm or too cool When nutrition is in short When information store is damaged (UV) When the competition and struggle for life is obviously Gene function ( 時間 ). 11

12. 只有在變動的狀況才看得見事物的真相 風 12 略

13. Change is a common phenomenon: Mutation and variation 13

14. Ways of change in bacteria Phenotypical adaptation Genetic changes Mutation  Spontaneous  Mutagens-induced  transposones Parasexual gene exchange  Transformation  Conjugation  Transduction 14

15. Mutant selection Selection by color, growth rate, nutrition dependence, drug resistance, morphology…. Selection in specific host, growth condition…. Timing of selection pressure 找到 mutants 的方法 ( 是事先發生 ? 還是因為誘發 ?) 15

16. Mutation Change in genetic material Most fatal, few may be beneficial (evolution) Base substitution: single base at one point in DNA sequence is replaced with a different base Frameshift mutation: DNA deletion or insertion Mutagen: Chemical:  Nitrous acid: remove an amino group from bases  Mustard gas: cross-links DNA strand  Nitrogen base analogs: compete with natural bases Radiation  Ionizing single or double DNA breaks  Ultraviolet: causes cross-links thymines to form dimers 類似物 ; 破壞結構 16

17. Concept of operon From left to right: Francois Jacob (1920- ), Jacques Monod (1910- 1976) and André Lwoff (1902-1994), awarded the Nobel Prize for Physiology or Medicine in 1965. Jacques Monod was director of the Pasteur Institute in Paris from 1971 to 1976. 17

18. Catabolic suppression Both the rate of synthesis and intracellular concentration of the enzymes need to metabolize lactose increase rapidly after lactose is added. When lactose is removed, synthesis of additional enzyme is repressed and soon stops altogether. The concentration of the remaining enzyme is then gradually diluted by cell growth 18

19. The lectose operon: the first regulation dogma by Jacob & Monod lacZ:  -galactosidase lacY: galactoside permease lacA: transactylase 沒有篩選突變的技術就做不了這個實驗，它還需要那兩種重要的技巧 : complementation and gene transfer 19 略

20. Gene exchange RequirementContact Genes transferred in most Transformation Free donor DNA fragment Competent recipient cell noMost capsule gene, unlimited Conjugation Sex pili on donor Fertility plasmid in donor Live cells Closely related species yes Drug resistance, resistant genes to toxin, enzymes etc. Transduction Donor lysed by phage Defective phage carries donor DNA Live competent recipient cell noToxins, drug resistance 20

21. Transformation ( 形質轉換 ) First reported by F. Griffith in 1928 on the infection of mice with s treptococcus pneumoniae (attempting to develop a vaccine), extensive studied by O.T. Avery in 1944. The pneumococcus transforming principle is DNA S triptococcus sp, Haemophilus influenzae, Bacillus sp can uptake DNA in nature Integrating into chromosome by recombination Some may require pili. Oswald Avery 21

22. What F. Griffith has found in 1928 Injection of a little S cells (pneumococcus) into a mouse will kill the mouse in 24 hours or so. But an injection of over 100 million (10 x 10 6 ) R cells is entirely harmless. smooth rough 22

23. Can it be an artifact? What Osward Avery has tested: enzymes to destroy all polysaccharides a lipase to destroy any lipids proteases to destroy all proteins RNase to destroy RNA DNase to destroy the DNA 23

24. Artificial transformation Cell fusion with protoplast/spheroplast CaCl 2 /heat shock Lipid (polyethylenglycol) Electroporation 24

25. 25 Laurenceau R. et al. 2013, A type IV pilus mediates DNA binding during natural transformation in Streptococcus pneumoniae. PLoS Pathog. 9(6):e1003473. Never to say never!

26. Conjugation Joshua Lederberg Found by J. Lederberg and E. Tatum in1946 Fertility factor is plasmid Direct contact required Can transfer to interspecies, intraspecies Application: gene map, plasmid typing 26

27. Conjugation 接合 27 See animation: http://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation6.htmlhttp://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation6.html 兩分多鐘

28. F factor: the plasmid 28 略

29. High-frequency recombination donors Plasmid integrated into chromosome More rapidly spread through the population (replicated along with bacteria chromosome) For E coli, the transfer of an entire chromosome takes about 100 min 29

30. The chromosome map is constructed 30

31. Transduction 性狀導入 First reported by N.D. Zinder and J. Lederber in 1952 in Samonella infected wit P22 phage (J Bacteriol 64:679) Mediated by phage Can be used as gene mapping 31

32. Generalized transduction - all regions of chromosome transduced, only chromosomal DNA (i.e., no phage DNA) in transducing particles. Specialized transduction - only specific regions of chromosome located near attachment site are transduced, transducing particles carry both chromosomal DNA and phage DNA. Two types of transduction 32

33.  Mobile genetic elements  No replication of its own  Mobilization is called transposition Transposon Barbara McClintock(1902-1992): Ds locus could change its position in the chromosome, 1948. 33 略

34. 虛枉社會的標準是： 1. 某號稱先進國家的名號 2. 使用科學語言的偽妝 2005/04/03 垃圾郵件的內容 34 裝肖ㄟ

35. Mutagens Reverse transcriptase DNA synthesis/sequence (DNA polymerase) Restriction enzymes Gene library (vectors) Southern blot, Northern blot, Western blot and Polymerase chain reaction. Ribozymes, RNAi Gene transfer: from transformation to transgenic animals The tools of recombinant DNA technology Engineering Detection Gene manipulation 35

36. 在微生物的遺傳進展之後 : A lot of imagination Decaffeinated coffee Antibody banana Vaccine tomato Growth hormone milk Xenotransplants ………. 36