Introduction of Biotechnology

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Oct 22, 2013 (3 years and 10 months ago)

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Graduate School of Engineering, Osaka University
Graduate School of Information Science, Osaka University
International Center for Biotechnology, Osaka University
Graduate School of Engineering, Osaka University
Graduate School of Information Science, Osaka University
International Center for Biotechnology, Osaka University
Introduction of Biotechnology
No.19: Microbial Genetics
Fundamentals
Introduction of Biotechnology
No.19: Microbial Genetics
Fundamentals
Handai Cyber University
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Hello! My name is Satoshi Harashima.
Scientific interest: Yeast Genetics, Yeast Genomics
Hobby: Listening and playing Jazz music
Date of birth: 21 May 1949 ; Birthplace: Ehime, Japan
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No.19 Microbial Genetics Fundamentals
I. Brief history of microbial genetics
II. Principle of genetic analyses
III. Yeast
genetics
IV. Bacterial genetics
V. Fungal
genetics
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I. Brief history of microbial genetics
•What is genetics
•Why do we study genetics?
•Advantage of classical genetics
•Feature of microbial materials
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What is genetics?
Geneticsis the scientific discipline to study physical and
molecular organization underlying hereditary process.
Why do children resemble their parents?
Geneticsis the study ofheredity
Heredityis the phenomenon whereby biological traits
are transmitted from one generation to another.
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Why do we study genetics?
Genetics occupies an important position in modern
biological sciences
Genetics is absolutely necessary for breeding of
organisms industrially used for biotechnology
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Major advantages of classical genetic approach
○Mutants can be isolated and characterized without
any a priori understanding of the molecular basis of
the function.
○To determine how many genes are involved in a
function?
○To find other genes whose products may interact
either physically or functionally with the products of
these genes.
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1865:Genetics begun with Mendel’s work
1900:Rediscovery of Mendel’s law
Plant
Untill1940:Fly and Corn as experimental
materials for genetic study
1940 〜:Microorganisms start to be used for
genetic study
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Microorganisms used for genetic studies
Escherichia coli
Neurosporacrass
Aspergillusnidulans
Saccharomycescerevisiae
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Feature of microbial
genetics
○Short generation time
○Accumulated knowledge for cultivation method
(suitable for Biochemistry)
○A large number of sample can be dealt with
(suitable for statistical analysis)
○Easy mutant-hunting due to haploidy
○Defined media can be used
(suitable for Biochemical genetics)
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Quiz 1
Do following characters give an advantage of
microorganisms in genetic study ?
1) Invisible cell by eyes
2) Short life span
3) Haploid vegitativecells
4) Large number of cells in small
space
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II. Principle of genetic analyses
III. Yeast genetics
•Isolation of mutants
•Dominance-recessivenesstest
•Complementation test
•Epistasis-hypostasis test
•Linkage analysis
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Essential approaches for genetic analyses
1) Isolation of mutants
2) Dominance-recessivenesstest
3) Complementation test(Recombination test)
4) Epistatic-hypostatic test
5) Linkage analysis
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Isolation of mutants
Genetics starts with isolation of mutants.
The most important step in genetic research.
his3-1: mutant allele:
Allele number
Gene nomenclature is different from organisms to organisms
Gene name (Three letter)
Locus number
Recessive mutation: lower caseDominant mutation: upper case
HIS3: wild type allele for Saccharomycescereivisiae
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Dominance-recessivenesstest
hisX
HISX
Mutant (His-)Wild type (His+)
Diploid
Cross
hisX/HISX
Mutation is Dominant
Mutation is Recessive
If His-phenotype
If diploid exhibits
If His+ phenotype
Loss of function?
Gain of function?
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Complementation test (for recessive mutation)
hisX
hisY
Mutant X (His-)Mutant Y (His-)
Diploid
Cross
Mutation occurs in different gene
Mutation occurs in the same gene
If diploid exhibits His-phenotype
hisX
hisY
hisX+
+ hisY
hisX
hisY
Mutant X (His-)Mutant Y (His-)
Diploid
Cross
If diploid exhibits His+phenotype
We could infer how many genes are involved
in a particular biological phenomenon.
hisX= hisYhisX≠hisY
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Epistasis-hypostasis test
phoX
phoY
Mutant (Uninducible
rAPaseproduction)
Mutant (Constitutive
rAPaseproduction)
Diploid
Cross
phoXphoY
Meiotic segregantharboring phoXphoYdouble
mutations
If phoXphoYsdouble mutant shows
phoXis epistatic
Constitutive phenotype
phoYis epistatic
Uninduciblephenotype
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Linkage analysis through meiosis
Deleted based on copyright concern.
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Linkage analysis
PDNPDT
HIS3 LEU2
HIS3 LEU2
his3 leu2
his3 leu2
his3 LEU2
his3 LEU2
HIS3 leu2
HIS3 leu2
HIS3 LEU2
HIS3 leu2
his3 LEU2
his3 leu2
Random assortment1: 1:4
Linkage>1:<1
Centromere linkage1:1:<4
Cross: HIS3 LEU2x his3 lue2
Spore 1
Spore 2
Spore 3
Spore 4
HIS3
his3
leu2
LUE2
1
2
3
4
Recombination
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100
1/2(T)
+
乐N
呯瑡T瑥瑲慤
=100
T
+
2乐N
(
)
2PD+NPD+T
()

100
1/2T

2乐N
(
)
[
]
+
4(NPD)
Totaltetrads
=100
T+6(NPD)
2(PD+NPD+T)
NPD type
his3 LEU2
his3 LEU2
HIS3 leu2
HIS3 leu2
T type
HIS3 LEU2
HIS3 leu2
his3 LEU2
his3 leu2
Map distance (cM) =
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Quiz 2
Which is a correct tetra-type tetrad from a cross
between MATahis3strain and MATαleu2
strain?
Spore A Spore B Spore C Spore D
1)His-Leu+, His+ Leu-, His+ Leu-, His-Leu+
2)His+Leu+, His-Leu+, His+Leu-, His-Leu-
3)His+ Leu+, His-Leu-, His+ Leu+, His-Leu-
4)His-Leu-, His-Leu+, His+ Leu-, His+ Leu-
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Five main genetic approaches
1) Isolation of mutants
2) Dominance-recessivenesstest
3) Complementation test(Recombination test)
4) Epistatic-hypostatic test
5) Linkage analysis
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Genetic processes to exchange genetic materials
in microorganisms
1) Sexual process (Conjugation, Mating )
Bacteria: E. coli
2) Parasexualprocess (hyphalfusion)
Bacteria: B. subtilis
Yeast : S. cerevisiae, S. pombe
3) Asexual processes (Transduction, Transformation)
Fungi: N.crassa, A. nidulans
Fungi: N.crassa, A. oryzae
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IV. Bacterial genetics
•Transformation
•Conjugation
•Transduction
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Brief history on bacterial transformation
1928: Griffith
Streptococcus pneumoniae
Pathogenic phenotype conversion
1944:Avery, MacLeod, McCarty
Transforming particle is DNA
1958: Spizizen
Natural transformation in Bacillus subtilis
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Bacterial transformation
Bacterial cell having an ability to incorporate DNA fragments
c
a
b
c
a
B
Transformant
X
X
Recombination
Uptake of DNA
fragments into cell
DNA
Competent cell
B
B
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1928: Griffith
Streptococcus pneumoniae
Pathogenic phenotype conversion
1944:Avery, MacLeod, McCarty
Transforming particle is DNA
1958: Spizizen
Natural transformation in Bacillus subtilis
1970: Mandel, Higa
Artificial transformation in Escherichia coli
Brief history on bacterial transformation
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Bacterial conjugation
E. coli cell transfers DNA segments
to another cell by direct cell-to-cell contact.
1946 (Lederbergand Tatum):
A
B
C
c
a
b
F factor
Escherichia coli
A
B
C
a
b
c
F factor origin
F factor terminus
a
B
c
Transfer during conjugation,
after integration
Recombination
Transconjugant
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Donor
Reciepient
Pilus
F plasmid
Conjugation bridge
E. colichromosome
(A)
(B)
F plasmid is transferred into recipient through a pore
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Transfer of chromosome is mediated byF
Integrated F
Donor
Recipient
Donor
Hfrstrain
(High frequency of recombination)
F plasmid
Conjugation
Recombination
X
X
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F-cells
do not contain the F factor and cannot transfer DNA
by conjugation. They are, however, recipients of DNA transferred from
F+, F’or Hfrcells by conjugation.
Summary of E. coliconjugation cycle
F+ cells
contain the F factor in the cytoplasm and can therefore
transfer F in a highly efficient manner to F-cells during conjugation.Hfrcells
have F integrated into the bacterial chromosome, not in the
cytoplasm.
F’cells
have F carrying a part of the bacterial chromosome, which is
generated from Hfrcell by pop-out.
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Transduction
1951 (Lederbergand Zinder):
Salmonella typhimurium
Bacteriophage
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a-
a+
a+
a+
a-
a+
Mechanism of general transduction in P1
Infection
Recombination
Transductant
Bacterial chromosome
Phage
a+
A phage containinga+ gene
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Phage λ
gal
bio
gal
bio
Integration between
galand biogene
λ
λ
gal+bio+
gal+bio+
Normal outlooping
λ
gal+
bio+
gal+
bio+
Rare abnormal outlooping
Mechanism of specialized transduction in phage λ
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Quiz 3
Gram-negative bacterium Agrobacterium
tumefaciencecan introduce a part of its Ti
plasmid DNA called T-DNA into plant cells with
cell-to-cell contact. What do you call this
phenomenon?
(1) Transformation
(2) Transduction
(3) Conjugation
(4) Transfection
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V. Fungalgenetics
•Sexual cycle (Mating)
•Parasexualcycle (Hyphalfusion)
•Complementation test using
heterokaryon
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Sexual and parasexualprocesses to exchange genetic materials
in fungi
Deleted based on copyright concern.
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Life cycle of
Neurosporacrassa,
the orange bread mold.
Deleted based on copyright concern.
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HyphaeFusion
Heterokaryon
Hyphaefuse to form heterokaryon
(n) yellow
(n) white
Spontaneous formation of diploid
(Green sectored colony)
(2n) parental
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(2n) parental
(n or 2n) yellow
(n or 2n) white
Yellow sectored colony
Diploid colony
White sectored colony
(w+/w y+/y)
Mitotic recombination or haploidizationoccurs
Deleted based on copyright
concern.
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arg-1
arg-2
Fusion
heterokaryongrows without arginine
Complementation test using heterokaryonin fungi
If two argmutations complements
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Graduate School of Engineering, Osaka University
Graduate School of Information Science, Osaka Universi
t
International Center for Biotechnology, Osaka Universit
y
Graduate School of Engineering, Osaka University
Graduate School of Information Science, Osaka Universi
t
International Center for Biotechnology, Osaka Universit
y
Handai Cyber University
No.19 : Microbial Genetics
Fundamentals
END
Handai Cyber University
No.19 : Microbial Genetics
Fundamentals
END
Thank you very much for your attention!
Don’t forget to check the next slide, please!
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Report (e-mail submition)
Q1. What is parasexualcycle observed in fungi.
Q2. What is the difference between generalized
transduction and specialized transduction?
Q3. What are advantages in using microorganisms
as experimental materials for genetics study?
Submit your report to “harashima@bio.eng.osaka-u.ac.jp”