Start with “what-and-how” questions (PR Murray, Medical Microbiology. 2013; 7 pages) Learning objective • Mutation, Repair, and Recombination. • Gene Exchanges in prokaryotic cells. • Mechanisms of Genetic transfer between cells. • Recombination. • Genetic Engineering. 很奇怪的排法 Traditional outline (R Bauman, Microbiology. 2007; 64 pages) • The structure and replication of genomes. • Gene function. • Mutation of genes. • Genetic recombination and transfer. • Recombinant DNA technology • Ethics and safety
There is nothing different between the basic structure of substances in livings carrying heritage Chromosome of an insect E. coli DNA spread George B. Kauffman, DNA Structure: Happy 50th Birthday! http://chemeducator.org/sbibs/s0008003/spapers/830219gk.htm
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)
略 Milestones in DNA History 化學 • 1869Johann 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. • 1928Franklin 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. • 1944Oswald Avery, Maclyn McCarty, and Colin MacLeod, identify Griffith's transforming agent as DNA. • 1949Erwin 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). • 1953James Watson and Francis Crick discover the molecular structure of DNA. 生物學 生化學 分子生物學 (物理)
略 Then, the day after 1953, the mystery of life becomes two coiled strands Rosalind Franklin (1920-1958)
Time 略 歷史問題:On the structure and replication (位置). • DNA replication starts from the origin: • E. coli temperature sensitive in initiation were synchronized by being transferred to 40oC for one hour, then shifted to 28oC. • At different times, the culture was pulse labeled with 3H-thymidine for 1 min
How to make sure DNA copies have been equally distributed into daughter cells? 略 歷史問題:On the structure and replication (如何分配). minicells
Gene function (生物化學的測量方法). Evidence of response to environmentheat shock responses of E. coli Protein synthesis: • Total proteins • Stimulated proteins • Other proteins
Gene function (時間). 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
略 只有在變動的狀況才看得見事物的真相 風
Change is a common phenomenon: Mutation and variation
Ways of change in bacteria • Phenotypical adaptation • Genetic changes • Mutation • Spontaneous • Mutagens-induced • transposones • Parasexual gene exchange • Transformation • Conjugation • Transduction
找到 mutants 的方法 (是事先發生?還是因為誘發?) Mutant selection • Selection by color, growth rate, nutrition dependence, drug resistance, morphology…. • Selection in specific host, growth condition…. • Timing of selection pressure
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
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.
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
略 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 The lectose operon:the first regulation dogma by Jacob & Monod lacZ: b-galactosidase lacY: galactoside permease lacA: transactylase
Transformation (形質轉換) • First reported by F. Griffith in 1928 on the infection of mice with streptococcus pneumoniae (attempting to develop a vaccine), extensive studied by O.T. Avery in 1944. • The pneumococcus transforming principle is DNA • Striptococcus sp, Haemophilus influenzae, Bacillus sp can uptake DNA in nature • Integrating into chromosome by recombination • Some may require pili. Oswald Avery
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 106) R cells is entirely harmless. smooth rough
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
Artificial transformation • Cell fusion with protoplast/spheroplast • CaCl2/heat shock • Lipid (polyethylenglycol) • Electroporation
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!
Joshua Lederberg Conjugation • 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
Conjugation 接合 See animation: http://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation6.html 兩分多鐘
略 F factor: the plasmid
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
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
Two types of transduction • 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.
略 TransposonBarbara McClintock(1902-1992): Ds locus could change its position in the chromosome, 1948. • Mobile genetic elements • No replication of its own • Mobilization is called transposition
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 裝肖ㄟ 2005/04/03 垃圾郵件的內容
The tools of recombinant DNA technology Engineering • 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 Detection Gene manipulation
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 • ……….