Transcript Microbial physiology

Microbial Genetics
楊倍昌
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Learning objective
Start with “what-and-how” questions (PR Murray, Medical Microbiology. 2013; 7 pages)
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Mutation, Repair, and Recombination.
Gene Exchanges in prokaryotic cells.
Mechanisms of Genetic transfer between
cells.
Recombination.
很奇怪的排法
Genetic Engineering.
Traditional outline (R Bauman, Microbiology. 2007; 64 pages)
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The structure and replication of genomes.
Gene function.
Mutation of genes.
Genetic recombination and transfer.
Recombinant DNA technology
Ethics and safety
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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
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 1866, Mendel published his lecture, a work that was
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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)
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化學
Milestones in DNA History
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生物學
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生化學
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分子生物學
(物理)
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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 speciesspecific. 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.
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Then, the day after 1953, the mystery of life
becomes two coiled strands
Rosalind Franklin (1920-1958)
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Replication by “conventional manner”
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歷史問題: On the structure and replication (位置).
 DNA replication starts
from the origin:
1. E. coli temperature
sensitive in initiation were
synchronized by being
transferred to 40oC for one
hour, then shifted to 28oC.
Time
2. At different times, the
culture was pulse labeled
with 3H-thymidine for 1
min
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歷史問題: On the structure and replication (如何分配).
How to make sure DNA copies
have been equally distributed
into daughter cells?
minicells
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Gene function (生物化學的測量方法).
Evidence of response to environment
heat shock responses of E. coli
Protein synthesis:
 Total proteins
 Stimulated proteins
○ Other proteins
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Gene function (時間).
Control, responses, adaptation
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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
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只有在變動的狀況才看得見事物的真相
風
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Change is a common phenomenon:
Mutation and variation
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Ways of change in bacteria
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Phenotypical adaptation
 Genetic changes
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Mutation
 Spontaneous
 Mutagens-induced
 transposones
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Parasexual gene exchange
 Transformation
 Conjugation
 Transduction
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找到 mutants 的方法 (是事先發生?還是因為誘發?)
Mutant selection
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Selection by color, growth rate, nutrition dependence,
drug resistance, morphology….
 Selection in specific host, growth condition….
 Timing of selection pressure
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Mutation
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Change in genetic material
 Most fatal, few may be beneficial (evolution)
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Base substitution: single base at one point in DNA sequence
is replaced with a different base
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Frameshift mutation: DNA deletion or insertion
Mutagen:
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類似物; 破壞結構
Chemical:
 Nitrous acid:
remove an amino group from bases
 Mustard gas: cross-links DNA strand
 Nitrogen base analogs: compete with natural bases
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Radiation
 Ionizing
single or double DNA breaks
 Ultraviolet: causes cross-links thymines to form dimers
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Concept of operon
From left to right:
Francois Jacob (1920- ),
Jacques Monod (19101976) 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.
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Catabolic suppression
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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
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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
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Gene exchange
Contact
Genes transferred
in most
no
Most capsule gene,
unlimited
 Sex pili on donor
 Fertility plasmid in
donor
 Live cells
 Closely related species
yes
Drug resistance,
resistant genes to
toxin, enzymes etc.
 Donor lysed by phage
 Defective phage carries
donor DNA
 Live competent
recipient cell
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Toxins, drug
resistance
Requirement
Transformation
Conjugation
Transduction
 Free donor DNA
fragment
 Competent recipient
cell
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Transformation (形質轉換)
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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
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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
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Can it be an artifact?
What Osward Avery has tested:
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enzymes to destroy all
polysaccharides
a lipase to destroy any lipids
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proteases to destroy all proteins
RNase to destroy RNA
DNase to destroy the DNA
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Artificial transformation
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Cell fusion with protoplast/spheroplast
CaCl2/heat shock
Lipid (polyethylenglycol)
Electroporation
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Never to say never!
Laurenceau R. et al. 2013, A type IV pilus mediates DNA binding
during natural transformation in Streptococcus pneumoniae.
PLoS Pathog. 9(6):e1003473.
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Conjugation
Joshua Lederberg
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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
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Conjugation 接合
See animation: http://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation6.html
兩分多鐘
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F factor: the plasmid
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High-frequency recombination donors
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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
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The chromosome map is
constructed
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Transduction 性狀導入
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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
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Two types of transduction
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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.
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Transposon
Barbara McClintock(1902-1992): Ds locus could change its
position in the chromosome, 1948.
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Mobile genetic elements
No replication of its own
Mobilization is called transposition
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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
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The tools of recombinant DNA technology
Engineering
Detection
Gene
manipulation
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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
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在微生物的遺傳進展之後: A lot of imagination
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Decaffeinated coffee
Antibody banana
Vaccine tomato
Growth hormone milk
Xenotransplants
……….
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