Chapter 8,Microbial Genetics
Ⅰ Hereditary material and its properties
Ⅱ Expression of genetic information
Ⅲ Mutation of genetic information in cells
Ⅳ Breeding by induced mutation and
genetic engineering
Genetics is the science of heredity; it is
concerned with the physical and chemical
properties of the hereditary material,how this
material is transmitted from one generation to
the next,and how the information it contains is
expressed during the development of an
individual,
---- Stanley R Maloy et al.,in
MICROBIAL GENETICS,1994
Microbial Genetics
- includes the heredity of some eukaryotes,all
prokaryotes and virus;
- introduces the structural characteristics of
hereditary materials,the expression and
regulation of genetic information,and the basic
rules of heredity and variation in microbes;
- introduces the basic strategies of controlling or
utilizing microbes by gene manipulation altering
the genetic information of microbes,
Ⅰ Hereditary material and its properties
A,The identification of hereditary
material
B,Deoxyribonucleic acid
C,Genome DNA and chromosomes
D,Hereditary genes beyond
chromosome
Expreiment 1,Ttransformation of S.pneumoniae
Fig 8,1（ a） Infection of S and R type cells
S type cell
R type cell
heat to kill
S type dead cell
dead
cell R type cell
Mixture of R cells & cell matters from S cells
fig 8.1（ b）
Cell extract R type cell
S type
cell
Saccharide
protein
Label
phage
T2 w/
32P or
35Ｓ
Infect
E coli
w/
labeled
T2
35Ｓ -Protein 32P-DNA
32P
35Ｓ
Experiment 2,Infection of phage T2
TMV
TMV HRV
Wild Separation Mixture Infection Isolation
HRV
Experiment 3,Reconstruction of viruses
Conclusion
The hereditary material of cellular
organisms is double stranded DNA,
The hereditary material of virus can be
single stranded or double stranded DNA
or RNA,i.e,ssDNA,dsDNA,ssRNA
or dsRNA,
B,Deoxyribonucleic acid
1,The composition and structure of
nucleic acid
2,The replication mode of DNA
3,The physical and chemical
properties of DNA
1,The composition and
structure of nucleic acid
Watson and Crick described DNA structure in 1953,
DNA most commomly occured as a double
helix,Two separated and antiparalled chains of DNA
are wound each other in a right-handed helical path,
with the sugar-phosphate backbones on the outside
and the bases,pared by hydrogen bonding and stacked
on each other,on the inside,A pares with T; G pares
with C,The two chains are complementary; one
specifies the sequence of the other,
Contributions of Rosalind Franklin
Watson and Crick proposed DNA structure
in 1953 by building models based on chemical
and physical data that had been gathered in
other labs,primarily x-ray diffraction data
collected by Rosalind Franklin et al,
2,Replication mode of DNA
? structure of bacteria DNA replication
DNA replication of eukaryote
10～ 100 mm
Template
New chain Origin
Replic,
forks Replic,forks
Fig 8,2
Nick
5’
5’
3’
3’
3’
Template
New chain
Complimentary chain
synthesis
Rolling circle mode of DNA replication
3,Physical and chemical properties of DNA
Stability of DNA
DNA solution is some stickiness,liable to the damage of
cut force,and is sensitive to nuclease,
In strong acid,DNA can be hydrolyzed into bases,
saccharides and phosphoric acid,
In watery alkalis e.g.0.2 M NaOH,double strand DNA
denaturalizes to single strand quickly,and single strand
DNA are destroyed further,
Specific hydrolysis
In specially watery acids (e.g.pH 3~4)，
indican bonds between different bases and
riboses can break specially,base sequence of
DNA can be determined according to this
special property,
Optical properties of DNA
Bases of DNA belong to chemicals of aroma
group,can absorb ultraviolet light (UV),
When concentration is 1 mg/ml，
absorbance at wavelength 260 nm is
dsDNA, A260=20
ssDNA,RNA, A260≌ 25
We determine the concentration of DNA by
A260,estimate the purity of DNA by A260 / A280
Heat denaturation of DNA
Double strand DNA can melt to produce single strand
DNA under certain high temperature,After double strands
completely melt,A260 can increase by 40％, The
temperature where A260 rises to the midpoint is defined as
melting temperature（ Tm）,
1.4
1.2
1.0
70 80 90
100° C Tm
A260
Renaturation,annealing or hybridization
When temperature drops slowly,single strand
DNA can pairs with each other,i.e,renaturation,
The base pairing of complimentary section of
DNA of different source is called annealing or
hybridization,this is widely applied in amplification
of DNA,site-directed induced mutation and gene
identification,
Molecular characteristics used in classification
and identification of microbes
The content of G＋ C in DNA is determined by Tm
generally,Among bacteria belonging to the same genus,the
variation of G＋ C content is commonly less than 10％。
The DNA－ DNA hybridization technology is used to
study microorganisms which are closely related,
If two strains are hybridized under optimal conditions
with relativity higher than 70%,and difference of Tm
values is less than 5％,they belong to the same species,
C,Genome and chromosome
Genome is referred to all the genes in a
bacterial cell or virus,and the haploid set of genes in
an eukariotic organism,
Chromosome is a structure consisting or
containing DNA which carries genetic information
essential to cells,Virus is not cellular organism,its
genes is not called chromosome,
D,Hereditary material beyond hromosome
DNA in mitochondria and chloroplasts can
replicate independently and can encode proteins
performing function of mitochondria and chloroplast，
it belongs to hereditary genes beyond chromosome,
Plasmids are commonly referred to hereditary
genes which can self-replicate independent of
chromosome,existing in bacterial and fungal cells,
Their sizes varies from 1 to 1000 kb,usually are
circular double strand DNA molecule,
II Expression of genetic information
A,Gene transcription and its regulation
of eukaryotes
B,Gene transcription and its regulation
of prokaryotes
C,Regulation during translation
D,Regulation signals and global
regulations
1,Gene transcription of eukaryotes
Common promoter is TATA box located in 25-30
base pairs upstream of initial transcription site,some
other genes contains a initial element overlapped by
initial transcription site,
RNA polymeraseⅡ catalyses and synthesizes Pre-
RNA,which must be processed to form mature mRNA。
Process of Pre-RNA proceeds in cell karyon,
Main procession steps,cap at 5′end,tail at 3′end,
wipe off introns by splicing and so on,
Occurring in the early stage of transcription of pre-
mRNA,when the length of newborn RNA reach 25～ 30
nucleotides,a enzyme hydrolyzes the phosphoric acid of
the first nucleotide (A or G) at 5′end of newborn pre-mRNA，
and adds 7-methyl –GTP to it,
2,Formation and structure of 5′cap
Fig 8.8 Structure of a cap,
CH3
3,Addition of poly-A tailing at 3′end
Adding poly-A at 3′end occurs before splicing introns,
In non-encoding region at the tail of Pre-mRNA,there is
a sequence of AAUAAA called poly-A site located about 20
nucleotides upristream of 3′end,
After special endonuclease recognizes the poly-A site，
it excises 20 nucleotides downstream of poly-A,
Then poly-A polymerase adds 20～ 250 A to the 3′end
to form poly-A tail。
Encoding sequences array in pre-
mRNA alternate with non-encoding
sequence, Then the non-encoding sequences
are called introns while encoding sequences
are called exons。
4,Excising introns by splicing
5,Regulation of gene transcription
of eukaryotes
Regulation of gene expression may
occur at any step such as transcription,
mRNA modification,stabilization,translation,
mRNA translation and degradation,
Regulation signals in eukaryotic cells
include 1) hormones and proteins,
2) environmantal factors,
B,Gene transcription and its regulation of
prokaryotes 1,Gene transcription in bacteria
Transcription and translation are coupled
Fig from reference 2
2,Transcription regulated by σ factors
The function of core enzyme of bacteria RNA
polymerase makes no difference to any gene,while σ factors
are the crucial subunit choosing transcription object,
Each kind of σ factor recognizes promoters with
specific sequences at the region of － 10 and － 35,
The region of － 10 and － 35 can not only recognized
by different σ factors,but also determine the intensity of
promoters,
e.g,E.coli
Under normal growth conditions,σ70 ( molecular mass
of 70 kDa) guides the activity of RNA polymerase,
When cell needs to make chemotactic move,it produces
σ28 ( 28 kDa) to start the synthesis of flagella and chemotactic
protein,
If environmental temperature rises suddenly,cells will
produce σ32 (32 kDa) to start the transcription of heat shock
proteins to protect cells from damage of heat,and to remove
denatured proteins,
3,Operon,and positive and negative
control of transcription
In prokaryotic cells,several structural genes close relative or
correlative to each other often arrange all together orderly,and can be
transcribed into the same RNA molecular,such a complete transcription unit
constituted by a transcription control region and one to a lot of structural
genes is called Operon,
Some genes can only express effectively after activated protein
combines to special site in DNA,The regulation pathway of gene
transcription activated or promoted by activated protein is called positive
control of transcription,
When repression proteins bind to special site of DNA,it exerts
repression or derepression function on transcription initiation,such
regulation pathway is called negative control of transcription,
e.g,Structure of lactose operon
Fig 8.6 There are two regulation genes,
（ 1） the binding site of repression rotein,that is Operator,lactose
participate in negative modulation,
（ 2） the binding site of activated protein CAP,cAMP participate
in positive modulation,
Reference 1
Attenuation terminates a transcription before it is
completed,Fig 8.7
4,Attenuation effect
Trp operon structure Sequence of premature
peptide
RNA polymerase RNA polymerase
ribosome ribosome
Abundant Trp in the environment Short of Trp in the environment
C,Regulation during translation
? Appearance frequency of rare codons
? Overlapped arrangement of genes
? SD sequence,distance from initial codon
? Secondary structure mRNA forms
? Existence of antisense RNA
Factors affecting translation
D,Regulation signals and global regulation
Microbes must respond to the
change of living conditions
instantly and compete with other
organisms or populations to obtain
and effectively use nutrients,
They have to produce,sense,
transmit signals,and have global
regulation over correlative multi-
operons at the same time,
1,Glucose effect and its mechanisms
When glucose,lactose,arabinose,
maltose exists together in the medium,E.coli
utilizes glucose first,Metabolization of other
saccharides do not start until glucose is used up,
Catabolite of glucose represses catabolite
of other sugar,this is known as catabolite
repression or glucose effect,
Catabolite repression mechanism
Cells uptake and metabolize large amount of
glucose followed by the decrease of the amount of
cAMP,this prevents the activation of lactose operon,
arabinose operon and maltose operon,which are
positively regulated by cAMP,
cAMP-CAP complex forms,binds to DNA，
and activats operons of other sugars when glucose is
in short,or cAMP is added to medium,
2,Stringent control
The content of ppGpp and pppGpp goes up rapidly
if bacteria can not get enough amino acid during their
growth,At the same time,the RNA synthesis and
accordingly protein synthesis slows down rapidly,thereby
cell is under conditions of slow metabolism and growth,
In the presence of enough amino acids,pppGpp and
ppGpp are degraded rapidly,synthesis of RNA and protein
is recovered quickly,This is a strategy for surviving in hard
time,named stringent control,
3,Quorum sensing system
Some bacteria possess a kind of regulation
system which can sense self cell density and make
relevant response to it,These bacteria release a
certain signal during growth,When cell density
reaches a critical level,signal molecules accumulate
to the concentration that can activate target operon,
such kind of regulationis called quorum sensing,
4,Two-component phosphorelay system
Two-component phosphorelay system is a kind of
modulation system that conducts signals through the
transfer of phosphoryl group to control gene transcription,
Two components,sensing protein kinase,
responding modulation protein,
Two-component system exists in bacteria widely,
and also exists in eukaryotic microbes,
Plants and animals also use the phosphorylating
mechanism to transduct signals,
Sporulation regulated by two-component
phosphorelay system in Bacillus subtilis
Fig8.8 σF and σE start sporulation,
Activate synthesis
of σF and σE
Prevent
synthesis of
AraB
III,Heredity and mutation of genetic
information in cells
A,Heredity and mutation of microorganisms
B,The molecular basis of mutation
C,Gene recombination
D,Gene transfer between prokaryotic cells
E,Gene transfer in Eukaryotic cells
A,Heredity and mutation of microorganisms
The classical fluctuation test is to observe self
mutation which is at low frequency,
Self mutation and induced mutation are essentially
the same,The genetic changes is caused by physical and
chemical factors acting on DNA,
The genetic changes in a few cells are capable of the
generation of new colony,
Auxotroph,drug resistant mutant,and temperature
sensitive mutant are common used in genetics,
E,coli fluctuation test of resistance to T1 Fig,8.1
Plate no,
Colony number resistant to T1 Plate no,Colony number resistant to T1
Different small tubes
variance
average
Different small tubes Repetition in large tubes Repetition in large tubes
Ames test
Auxotroph
Auxotrophic plate
.,,
.,,
,
,
:,¨,,..,,
, ;, ;.,
,¨,,
,
:,¨
.,.
.
,.
, ;
, ;
.,
.¨
,,.,,
.,
,
Testing
reagent
(Fig,8.10)
Normal revert mutation
Mutation
Ames test is a standard method to test the mutation ability of chemicals,It
depends on whether the testing reagent increase reversion of an auxotroph,
B,The molecular basis of mutation
The abnormal changes in the DNA structure
resulted from various agents is called DNA
damage,
DNA damage causes gene mutation or cell
death,
DNA molecules mismatch by self action,In
addition,the molecular basis of mutation is DNA
damage of physical or chemical mutagens,
Chemical and physical mutagens
Physical mutagens
Mutagens ionizing radiation,UV
Chemical mutagens
deaminating agents,
alkylating agents,base analogs,
insertion agent,cross linking agent,
Mutagens and mutation Fig,7.11
(a) Deamination by nitrous acid ；
(b) Alkylating agent and methylated nuleoside；
(c) 5-Bromodeoxy-uridine pairs to gunine,
Generation of pyrimidine derivatives by UV
Fig,7.12
thymine
cytosine
cytosine hydrate thymine dimer
thymine-cytosine dimer dihydrothymine
UVL
Molecule mutation and information variety
Silent mutation
Missense mutation
Nonsense mutation
Frame-shift mutation
Elongtion mutation
Codon mutation
C,Gene recombination
Homologous recombination is known as generalized gene
recombination in biology,It occurs in any pair of DNA molecules having
homologous sequence at any position which is RecA-dependant,
Site-specific recombination depends on limited sequence
similarity between the recombining DNAs,It requires specific DNA
sequence in donor and target molecules which is recognized and
catalyzed by specific protein factor,
Transposon,a DNA element moving from one DNA address to
another,Transposition is a DNA recombination resulted by transposons
without recA function and DNA homology,The transposon contains
inverted terminal repeats that can be recognized and cut by Transposase,
Integration of l phage into E,coli host DNA at specific
site (a) and the excision of the phage DNA out again during
prophage induction (b),
Site-specific recombination Fig,8.14
Transposon generation and insertion of direct repeats in
host DNA flanking a transposon,
Transposition Fig,8.15
Target sequence Transposon
D,Gene transfer between prokaryotic cells
Conjugation is a transfer of a plasmid from a
donor cell to a receptor cell when the they joints,
Transformation refers to the cell adoption of
foreign DNA from environment,which changes
corresponding genotype or phenotype of receptors,
Transduction refers to the phage-mediate
DNA transfer from a donor microbe to a receptor
microbe,
E,Gene transfer in eukaryotic cells
Parasexual and sexual reproduction Fig,8.2
Charaters Parasexual reproduction Sexual reproduction
Combined parent cells
Self survival of heterocaryon
Cell morphology after nucleus
infusion
Method of haploid formation
from diploid
Frequency of combination
Somatic cells of same shape Somatic cells of special differentiation
yes yes or no
Same to haploid Different from haploid
Karyokinesis Meiosis
Low High
Ⅳ Breeding by induced mutation and
genetic engineering
A,Breeding by induced mutation
B,Breakthrough leading to recombinant
DNA technology
C,Genetic engineering
D,Geneic modification of organisms,gene
therapy and genetic medicines
UVL VL
Photolyase
UV Damage & photoreactivation of DNA
EXAMPLE
Procedures for UV mutagenesis
Induced mutation of
Penicillium chrysogenum
Breeding of strain NRRL-B25 for production of penicillin
Strain Mutagen Yield (U/ml) Time
NRRL-B25 250 1943
NRRL-X1612 X-rays 500 1943
NRRL-Q176 UV light 850 1945
NRRL-WIS-47-1564 UV light 850 1947
--- --- 50000 1977
? It contributes greatly to early biotechnology,
? Industrial strain can be improved to a good
level before related genetic information is obtained,
? Relatively minor modifications can be obtained,
? The nature of mutations is not known,
? No new function obtained without
corresponding gene,
The advantages and disadvantages of
breeding by induced mutation
B,Breakthroughs in
recombinant DNA technology
1,Restriction enzymes
2,Reverse transcriptase
3,DNA ligase
4,DNA polymerase and Polymerase Chain
Reaction
1,Restriction enzymes
In 1970,Arber,W,& H,Smith reported
microbial enzymes cut dsDNA at specific site,
These are called restriction enzymes,The discovery
indicated the first step in rDNA technology,
Restriction enzymes recognize 4 ～ 8 bases
of palindrome sequences,
Fig,7.18.(a)
Restriction enzyme
digestion of DNA
Cut to produce
Sticky ends,
or
Blunt ends,
XhoI
SciI
Fig,7.8,（ b）,（ c）
2,Reverse transcriptase
Reverse transcriptase is an RNA-
dependent DNA polymerase that uses RNA as a
template to form a DNA copy,In 1970,reverse
transcriptase is found in retroviruses to from a
DNA copy from a RNA genome template,
It synthesis single-strand cDNA from
5′→3 in the presence of a primer,a template
molecule,dNTP and Mg2+,
3,DNA ligases
DNA ligases creates a phosphodiester bond
between adjacent 3'-hydroxylated and 5'-
phosphorylated termini in duplex DNA structures,
In 1972,David Jackson et,al annealed sticky
ends of DNA fragments,followed by ligation using
DNA ligases,Thus ligation technology between
vectors and foreign DNA applied in gene cloning is
developed,and recombinant DNA molecule is
obtained,
4,DNA polymerase and polymerase
chain reaction
DNA polymerase is an enzyme that catalyzes template-
dependent synthesis of DNA from its deoxyribonucleoside 5‘-
triphosphate precursors,
In 1985,Kary Mullis et al,creates Polymerase Chain
Reaction (PCR) using DNA polymeras,It amplifies a specific
DNA fragment up to millions copies in vitro in a short period
of time,
The inventor wins Nobel Prize for the great contribution
that PCR brings to modern biotechnology,
Heat-stable DNA polymerase
from extremophiles
e.g,Taq polymerase from Thermus aquatcus
Half-life > 2 h at 95?C
Allow to perform in machine,thermal cycler
T (?C)
90
70
50
Time (min)
Cycle 1 Cycle 2 Cycle 3 Cycle
C,Genetic engineering
1,The overall strategy of genetic
engineering
2,Cloning and characterization of
target genes
3,Host and vector
4,Production of recombinant protein
1,The overall strategy of genetic
engineering (1)
A group of techniques for manipulating
DNA outside the organism from which it was
obtained,and reintroducing the recombinant or
modified DNA into another cell where it will
exert its effects,
Blue-white screening
Activity screening
E,coli expression frame
Lac promotor
RBS Ribosome binding site
ATG starting codon
MCS Multiple cloning sites
LacZ’ sequence for blue-white screening
Selective mark
E,coli replicon
Phosphorimaging
or color reaction
32P-labled probe
DIG-labled probe and marker
3,Host and vector
Cloning vector is a DNA molecule
that can replicate,and is used to transport a
piece of inserted foreign DNA into a
recipient cell,It may be a plasmid,phage,
cosmid or artificial chromosome,
----- L M Prescott et al.,in
MICROBIOLOGY,5th ed,
Vectors carry genes
into host cells
Recombination of plasmid and foreign gene
An example of a plasmid
E,coli K12 and derived strains
E,coli K12
Fe transportor
Antigen O
Toxin Capsule
? Small genome
? No plsmid
? Acapsule
? No fimbra adhesin
Fimbra
adhesin
? Antigen O reduction
? No toxin
? No Fe transportor
Wide cell
Advantages in production of recombinant
protein
a,Use of modified gene;
b,Increase of gene copies;
c,Strong promoter for transcription;
d,Optimized translation;
e,Optional regulation;
f,Easy cultivation,
4,Production of recombinant protein
a,Use of modified gene
YYYYYY
XXXXXX
YYYAAATTT CCCAAAYYY
XXXTTTAAA GGGTTTXXX
Blunting Kination
Ligation PCR
b.,c,& d,In central dogma
? lac Promoter from
E.coli
? Recombinant of lac &
trp,Ptac
? Promoter from phage
T7
? PL from E.coli phage l
? PHsh from E.coli
Induction of expression in E,coli
e,Optional regulation
PL +1 RBS FG Terminator
30℃
40 ℃
mRNA
Heat shock * ts repressor
* A temperature sensitive protein encoded by mutant cI857(ts)
Induction of gene expression in PL vector
T7 Promoter
FG
Host cell Chromosome
Vector
vector
T7 RNA
polymerase
Gene expression controlled by T7 promoter
Plac/PL
f,Easy cultivation
D,Genetic modification of organisms,gene
therapy and genetic medicines
1,Recombinant microorganisms
2,Transgenic plants
3,Transgenic animals
4,Gene therapy and genetic medicines
Definition
Metabolic (pathway) engineering
is the use of molecular techniques (or
recombinant DNA techniques) to improve
the efficiency of pathways that synthesize
specific products,
----- L M Prescott et al.,in
MICROBIOLOGY,5th ed
1,Recombinant microorganisms
Fermentation of ethanol by Zymomonas
mobilis
Entner-Doudoroff pathway
Pyruvate
Acetaldehyde + CO2
Ethanol
Pyruvate
decarboxylase
Alcohol dehydrogenase
E,coli 的代谢工程
EMP途径
乳酸 丙酮酸 乙醛
甲酸 乙醇脱氢酶
CO2+H2 乙酰辅酶 A 乙醇
磷酸转移酶 乙醛脱氢酶
乙酰磷酸 乙醛
乙酸激酶 乙醇脱氢酶
乙酸 乙醇
乳酸脱氢酶 丙酮酸脱羧酶
乙醇操纵子设计
宿主菌株的选择
基因的整合
表达水平的筛选
条件优化等
乙醇发酵的代谢工程
Production of ethanol from corn fiber hydrolysate
Strain Yield Recovery Rate
（ g/l） （ %） （ g/l /h）
Sacharomyces 1400 21.0 98 1.60
Z,mobilis CP4 22.6 88 1.04
E,coli KO11 34.7 80 1.16
EXAMPLE
Metabolic engineered strains
2,Transgenic plants
Agrobacterium spp,living in earth infects plant
cells through callus with transference of T-DNA to a
plant gnome,Thus the plant cells produce growth
regulators,and synthesize nutrition for A,spp,
A,tumefaciens cause plant cell overgrowth to
form cell tumor that forms crown gall ； A,rhizogenes
cause abnormal growth of roots cells to form,hairy
roots”,They are important tools of plant genentic
engineering,
T-DNA
intergration
Secretion
Cytopharynx
Auxin
Plant cell
Nuclear
pore
Opine
Damage
Secretion
Acetosyringon
Agrobacterium sp,
Nucleus
Pi-VirA
Induction of vir
expression
T-DNA from
Ti plasmid
VirD2 VirB
Agrobacterium tumefaciens and plants
3,Transgenic animals
Most efficient method,
Transduction of animal or animal embryo by virus,
Main purpose,
? Production of human tissue or apparatus
? Construction of animal models for human disease therapy,
? Insects,pigs,sheep,and et al,can be applied in human
protein expression,
Retrovirus
Single-strand RNA that infects mammalia;
Double-strand DNA is synthesized by RNA
reverse transcriptation after infection;
It is a ideal vector for gene therapy because
of the strong promotor,
Baculovirus
It is a insect virus of rod particles containing
double-strand DNA,which is packed up by
polyhedrin protein in vivo or expelled,
? The foreign gene can be overexpressed controlled
by the strong promoter of polyhedrin protein;
? Insect system completed the post-translation
modification;
? It is suitable for animal protein production,
4,Gene therapy and genetic medicines
Some disease is caused by gene dysfuction,
In gene therapy,a nonfunctional or dysfunctional
gene is augmented or replaced by a functional gene,
Retrovirus is always used as a vector in
gene therapy,which introduce new gene into
human multiplication cells,
Antisense RNA as genetic medicine
Some diseases are caused by abnormal
expression of a certain protein,such as most virus
disease,cancer,Alzheimer’s disease,They can be
efficiently prevented by reduction of transcription
and expression of corresponding genes,
Antisense RNA complements the sequence in
the translation initiation region of mRNA,that
efficiently stops the ribosome combination and
translation initiation,
RNA interference
RNA interference,first observed in petunias,is a
phenomenon of gene silencing at the mRNA level
offering a quick and easy way to determine the function
of a gene both in vivo and in vitro,
Duplexes of 21-23 nt RNAs (small-interfering
RNA,siRNA) produced by RNase in cytoplasm are
introduced into a cell where they associate with specific
proteins into a multi-protein siRNA complex (RNA
induced silencing complex,RISC),RISC then binds to,
and degrades the target mRNA in the cell in a highly
specific manner,
siRNA as genetic medicine
siRNA has advantages over antisense
RNA in the efficiency and specialization,
siRNA is widely applied in the clinic
therapy of eye disease and hepatitis,
It was reported in 2005 the scientists in
our country succeeded in retaining the SARS
by siRNA,
References
1,Madigan,MT,JM Martinko,and J Parker,2003,Brock Biology of
Microbiology,10th ed,Printice Hall,New Jersey,
2,Talaro,KP,2005,Foundations in Microbiology,5th ed,McGraw-Hill
co,& High Education Press,Beijing,
3,Prescott,LM,JP Harley,and DA Klein,2002,Microbiology,5th ed,
McGraw-Hill co,& High Education Press,Beijing,
4,Turner,PC,AG McLennan,AD Bates and MRH White,1997,Instant
Notes in Molecular Biolgy,Bios Scentific Publishers Limited,2000,
5,Maloy,SR,JE Cronan Jr,and D Freifelder,1994,Microbial Genetics,
2nd ed,Jones and Bartlett Publishers,Boston,
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Ⅰ Hereditary material and its properties
Ⅱ Expression of genetic information
Ⅲ Mutation of genetic information in cells
Ⅳ Breeding by induced mutation and
genetic engineering
Genetics is the science of heredity; it is
concerned with the physical and chemical
properties of the hereditary material,how this
material is transmitted from one generation to
the next,and how the information it contains is
expressed during the development of an
individual,
---- Stanley R Maloy et al.,in
MICROBIAL GENETICS,1994
Microbial Genetics
- includes the heredity of some eukaryotes,all
prokaryotes and virus;
- introduces the structural characteristics of
hereditary materials,the expression and
regulation of genetic information,and the basic
rules of heredity and variation in microbes;
- introduces the basic strategies of controlling or
utilizing microbes by gene manipulation altering
the genetic information of microbes,
Ⅰ Hereditary material and its properties
A,The identification of hereditary
material
B,Deoxyribonucleic acid
C,Genome DNA and chromosomes
D,Hereditary genes beyond
chromosome
Expreiment 1,Ttransformation of S.pneumoniae
Fig 8,1（ a） Infection of S and R type cells
S type cell
R type cell
heat to kill
S type dead cell
dead
cell R type cell
Mixture of R cells & cell matters from S cells
fig 8.1（ b）
Cell extract R type cell
S type
cell
Saccharide
protein
Label
phage
T2 w/
32P or
35Ｓ
Infect
E coli
w/
labeled
T2
35Ｓ -Protein 32P-DNA
32P
35Ｓ
Experiment 2,Infection of phage T2
TMV
TMV HRV
Wild Separation Mixture Infection Isolation
HRV
Experiment 3,Reconstruction of viruses
Conclusion
The hereditary material of cellular
organisms is double stranded DNA,
The hereditary material of virus can be
single stranded or double stranded DNA
or RNA,i.e,ssDNA,dsDNA,ssRNA
or dsRNA,
B,Deoxyribonucleic acid
1,The composition and structure of
nucleic acid
2,The replication mode of DNA
3,The physical and chemical
properties of DNA
1,The composition and
structure of nucleic acid
Watson and Crick described DNA structure in 1953,
DNA most commomly occured as a double
helix,Two separated and antiparalled chains of DNA
are wound each other in a right-handed helical path,
with the sugar-phosphate backbones on the outside
and the bases,pared by hydrogen bonding and stacked
on each other,on the inside,A pares with T; G pares
with C,The two chains are complementary; one
specifies the sequence of the other,
Contributions of Rosalind Franklin
Watson and Crick proposed DNA structure
in 1953 by building models based on chemical
and physical data that had been gathered in
other labs,primarily x-ray diffraction data
collected by Rosalind Franklin et al,
2,Replication mode of DNA
? structure of bacteria DNA replication
DNA replication of eukaryote
10～ 100 mm
Template
New chain Origin
Replic,
forks Replic,forks
Fig 8,2
Nick
5’
5’
3’
3’
3’
Template
New chain
Complimentary chain
synthesis
Rolling circle mode of DNA replication
3,Physical and chemical properties of DNA
Stability of DNA
DNA solution is some stickiness,liable to the damage of
cut force,and is sensitive to nuclease,
In strong acid,DNA can be hydrolyzed into bases,
saccharides and phosphoric acid,
In watery alkalis e.g.0.2 M NaOH,double strand DNA
denaturalizes to single strand quickly,and single strand
DNA are destroyed further,
Specific hydrolysis
In specially watery acids (e.g.pH 3~4)，
indican bonds between different bases and
riboses can break specially,base sequence of
DNA can be determined according to this
special property,
Optical properties of DNA
Bases of DNA belong to chemicals of aroma
group,can absorb ultraviolet light (UV),
When concentration is 1 mg/ml，
absorbance at wavelength 260 nm is
dsDNA, A260=20
ssDNA,RNA, A260≌ 25
We determine the concentration of DNA by
A260,estimate the purity of DNA by A260 / A280
Heat denaturation of DNA
Double strand DNA can melt to produce single strand
DNA under certain high temperature,After double strands
completely melt,A260 can increase by 40％, The
temperature where A260 rises to the midpoint is defined as
melting temperature（ Tm）,
1.4
1.2
1.0
70 80 90
100° C Tm
A260
Renaturation,annealing or hybridization
When temperature drops slowly,single strand
DNA can pairs with each other,i.e,renaturation,
The base pairing of complimentary section of
DNA of different source is called annealing or
hybridization,this is widely applied in amplification
of DNA,site-directed induced mutation and gene
identification,
Molecular characteristics used in classification
and identification of microbes
The content of G＋ C in DNA is determined by Tm
generally,Among bacteria belonging to the same genus,the
variation of G＋ C content is commonly less than 10％。
The DNA－ DNA hybridization technology is used to
study microorganisms which are closely related,
If two strains are hybridized under optimal conditions
with relativity higher than 70%,and difference of Tm
values is less than 5％,they belong to the same species,
C,Genome and chromosome
Genome is referred to all the genes in a
bacterial cell or virus,and the haploid set of genes in
an eukariotic organism,
Chromosome is a structure consisting or
containing DNA which carries genetic information
essential to cells,Virus is not cellular organism,its
genes is not called chromosome,
D,Hereditary material beyond hromosome
DNA in mitochondria and chloroplasts can
replicate independently and can encode proteins
performing function of mitochondria and chloroplast，
it belongs to hereditary genes beyond chromosome,
Plasmids are commonly referred to hereditary
genes which can self-replicate independent of
chromosome,existing in bacterial and fungal cells,
Their sizes varies from 1 to 1000 kb,usually are
circular double strand DNA molecule,
II Expression of genetic information
A,Gene transcription and its regulation
of eukaryotes
B,Gene transcription and its regulation
of prokaryotes
C,Regulation during translation
D,Regulation signals and global
regulations
1,Gene transcription of eukaryotes
Common promoter is TATA box located in 25-30
base pairs upstream of initial transcription site,some
other genes contains a initial element overlapped by
initial transcription site,
RNA polymeraseⅡ catalyses and synthesizes Pre-
RNA,which must be processed to form mature mRNA。
Process of Pre-RNA proceeds in cell karyon,
Main procession steps,cap at 5′end,tail at 3′end,
wipe off introns by splicing and so on,
Occurring in the early stage of transcription of pre-
mRNA,when the length of newborn RNA reach 25～ 30
nucleotides,a enzyme hydrolyzes the phosphoric acid of
the first nucleotide (A or G) at 5′end of newborn pre-mRNA，
and adds 7-methyl –GTP to it,
2,Formation and structure of 5′cap
Fig 8.8 Structure of a cap,
CH3
3,Addition of poly-A tailing at 3′end
Adding poly-A at 3′end occurs before splicing introns,
In non-encoding region at the tail of Pre-mRNA,there is
a sequence of AAUAAA called poly-A site located about 20
nucleotides upristream of 3′end,
After special endonuclease recognizes the poly-A site，
it excises 20 nucleotides downstream of poly-A,
Then poly-A polymerase adds 20～ 250 A to the 3′end
to form poly-A tail。
Encoding sequences array in pre-
mRNA alternate with non-encoding
sequence, Then the non-encoding sequences
are called introns while encoding sequences
are called exons。
4,Excising introns by splicing
5,Regulation of gene transcription
of eukaryotes
Regulation of gene expression may
occur at any step such as transcription,
mRNA modification,stabilization,translation,
mRNA translation and degradation,
Regulation signals in eukaryotic cells
include 1) hormones and proteins,
2) environmantal factors,
B,Gene transcription and its regulation of
prokaryotes 1,Gene transcription in bacteria
Transcription and translation are coupled
Fig from reference 2
2,Transcription regulated by σ factors
The function of core enzyme of bacteria RNA
polymerase makes no difference to any gene,while σ factors
are the crucial subunit choosing transcription object,
Each kind of σ factor recognizes promoters with
specific sequences at the region of － 10 and － 35,
The region of － 10 and － 35 can not only recognized
by different σ factors,but also determine the intensity of
promoters,
e.g,E.coli
Under normal growth conditions,σ70 ( molecular mass
of 70 kDa) guides the activity of RNA polymerase,
When cell needs to make chemotactic move,it produces
σ28 ( 28 kDa) to start the synthesis of flagella and chemotactic
protein,
If environmental temperature rises suddenly,cells will
produce σ32 (32 kDa) to start the transcription of heat shock
proteins to protect cells from damage of heat,and to remove
denatured proteins,
3,Operon,and positive and negative
control of transcription
In prokaryotic cells,several structural genes close relative or
correlative to each other often arrange all together orderly,and can be
transcribed into the same RNA molecular,such a complete transcription unit
constituted by a transcription control region and one to a lot of structural
genes is called Operon,
Some genes can only express effectively after activated protein
combines to special site in DNA,The regulation pathway of gene
transcription activated or promoted by activated protein is called positive
control of transcription,
When repression proteins bind to special site of DNA,it exerts
repression or derepression function on transcription initiation,such
regulation pathway is called negative control of transcription,
e.g,Structure of lactose operon
Fig 8.6 There are two regulation genes,
（ 1） the binding site of repression rotein,that is Operator,lactose
participate in negative modulation,
（ 2） the binding site of activated protein CAP,cAMP participate
in positive modulation,
Reference 1
Attenuation terminates a transcription before it is
completed,Fig 8.7
4,Attenuation effect
Trp operon structure Sequence of premature
peptide
RNA polymerase RNA polymerase
ribosome ribosome
Abundant Trp in the environment Short of Trp in the environment
C,Regulation during translation
? Appearance frequency of rare codons
? Overlapped arrangement of genes
? SD sequence,distance from initial codon
? Secondary structure mRNA forms
? Existence of antisense RNA
Factors affecting translation
D,Regulation signals and global regulation
Microbes must respond to the
change of living conditions
instantly and compete with other
organisms or populations to obtain
and effectively use nutrients,
They have to produce,sense,
transmit signals,and have global
regulation over correlative multi-
operons at the same time,
1,Glucose effect and its mechanisms
When glucose,lactose,arabinose,
maltose exists together in the medium,E.coli
utilizes glucose first,Metabolization of other
saccharides do not start until glucose is used up,
Catabolite of glucose represses catabolite
of other sugar,this is known as catabolite
repression or glucose effect,
Catabolite repression mechanism
Cells uptake and metabolize large amount of
glucose followed by the decrease of the amount of
cAMP,this prevents the activation of lactose operon,
arabinose operon and maltose operon,which are
positively regulated by cAMP,
cAMP-CAP complex forms,binds to DNA，
and activats operons of other sugars when glucose is
in short,or cAMP is added to medium,
2,Stringent control
The content of ppGpp and pppGpp goes up rapidly
if bacteria can not get enough amino acid during their
growth,At the same time,the RNA synthesis and
accordingly protein synthesis slows down rapidly,thereby
cell is under conditions of slow metabolism and growth,
In the presence of enough amino acids,pppGpp and
ppGpp are degraded rapidly,synthesis of RNA and protein
is recovered quickly,This is a strategy for surviving in hard
time,named stringent control,
3,Quorum sensing system
Some bacteria possess a kind of regulation
system which can sense self cell density and make
relevant response to it,These bacteria release a
certain signal during growth,When cell density
reaches a critical level,signal molecules accumulate
to the concentration that can activate target operon,
such kind of regulationis called quorum sensing,
4,Two-component phosphorelay system
Two-component phosphorelay system is a kind of
modulation system that conducts signals through the
transfer of phosphoryl group to control gene transcription,
Two components,sensing protein kinase,
responding modulation protein,
Two-component system exists in bacteria widely,
and also exists in eukaryotic microbes,
Plants and animals also use the phosphorylating
mechanism to transduct signals,
Sporulation regulated by two-component
phosphorelay system in Bacillus subtilis
Fig8.8 σF and σE start sporulation,
Activate synthesis
of σF and σE
Prevent
synthesis of
AraB
III,Heredity and mutation of genetic
information in cells
A,Heredity and mutation of microorganisms
B,The molecular basis of mutation
C,Gene recombination
D,Gene transfer between prokaryotic cells
E,Gene transfer in Eukaryotic cells
A,Heredity and mutation of microorganisms
The classical fluctuation test is to observe self
mutation which is at low frequency,
Self mutation and induced mutation are essentially
the same,The genetic changes is caused by physical and
chemical factors acting on DNA,
The genetic changes in a few cells are capable of the
generation of new colony,
Auxotroph,drug resistant mutant,and temperature
sensitive mutant are common used in genetics,
E,coli fluctuation test of resistance to T1 Fig,8.1
Plate no,
Colony number resistant to T1 Plate no,Colony number resistant to T1
Different small tubes
variance
average
Different small tubes Repetition in large tubes Repetition in large tubes
Ames test
Auxotroph
Auxotrophic plate
.,,
.,,
,
,
:,¨,,..,,
, ;, ;.,
,¨,,
,
:,¨
.,.
.
,.
, ;
, ;
.,
.¨
,,.,,
.,
,
Testing
reagent
(Fig,8.10)
Normal revert mutation
Mutation
Ames test is a standard method to test the mutation ability of chemicals,It
depends on whether the testing reagent increase reversion of an auxotroph,
B,The molecular basis of mutation
The abnormal changes in the DNA structure
resulted from various agents is called DNA
damage,
DNA damage causes gene mutation or cell
death,
DNA molecules mismatch by self action,In
addition,the molecular basis of mutation is DNA
damage of physical or chemical mutagens,
Chemical and physical mutagens
Physical mutagens
Mutagens ionizing radiation,UV
Chemical mutagens
deaminating agents,
alkylating agents,base analogs,
insertion agent,cross linking agent,
Mutagens and mutation Fig,7.11
(a) Deamination by nitrous acid ；
(b) Alkylating agent and methylated nuleoside；
(c) 5-Bromodeoxy-uridine pairs to gunine,
Generation of pyrimidine derivatives by UV
Fig,7.12
thymine
cytosine
cytosine hydrate thymine dimer
thymine-cytosine dimer dihydrothymine
UVL
Molecule mutation and information variety
Silent mutation
Missense mutation
Nonsense mutation
Frame-shift mutation
Elongtion mutation
Codon mutation
C,Gene recombination
Homologous recombination is known as generalized gene
recombination in biology,It occurs in any pair of DNA molecules having
homologous sequence at any position which is RecA-dependant,
Site-specific recombination depends on limited sequence
similarity between the recombining DNAs,It requires specific DNA
sequence in donor and target molecules which is recognized and
catalyzed by specific protein factor,
Transposon,a DNA element moving from one DNA address to
another,Transposition is a DNA recombination resulted by transposons
without recA function and DNA homology,The transposon contains
inverted terminal repeats that can be recognized and cut by Transposase,
Integration of l phage into E,coli host DNA at specific
site (a) and the excision of the phage DNA out again during
prophage induction (b),
Site-specific recombination Fig,8.14
Transposon generation and insertion of direct repeats in
host DNA flanking a transposon,
Transposition Fig,8.15
Target sequence Transposon
D,Gene transfer between prokaryotic cells
Conjugation is a transfer of a plasmid from a
donor cell to a receptor cell when the they joints,
Transformation refers to the cell adoption of
foreign DNA from environment,which changes
corresponding genotype or phenotype of receptors,
Transduction refers to the phage-mediate
DNA transfer from a donor microbe to a receptor
microbe,
E,Gene transfer in eukaryotic cells
Parasexual and sexual reproduction Fig,8.2
Charaters Parasexual reproduction Sexual reproduction
Combined parent cells
Self survival of heterocaryon
Cell morphology after nucleus
infusion
Method of haploid formation
from diploid
Frequency of combination
Somatic cells of same shape Somatic cells of special differentiation
yes yes or no
Same to haploid Different from haploid
Karyokinesis Meiosis
Low High
Ⅳ Breeding by induced mutation and
genetic engineering
A,Breeding by induced mutation
B,Breakthrough leading to recombinant
DNA technology
C,Genetic engineering
D,Geneic modification of organisms,gene
therapy and genetic medicines
UVL VL
Photolyase
UV Damage & photoreactivation of DNA
EXAMPLE
Procedures for UV mutagenesis
Induced mutation of
Penicillium chrysogenum
Breeding of strain NRRL-B25 for production of penicillin
Strain Mutagen Yield (U/ml) Time
NRRL-B25 250 1943
NRRL-X1612 X-rays 500 1943
NRRL-Q176 UV light 850 1945
NRRL-WIS-47-1564 UV light 850 1947
--- --- 50000 1977
? It contributes greatly to early biotechnology,
? Industrial strain can be improved to a good
level before related genetic information is obtained,
? Relatively minor modifications can be obtained,
? The nature of mutations is not known,
? No new function obtained without
corresponding gene,
The advantages and disadvantages of
breeding by induced mutation
B,Breakthroughs in
recombinant DNA technology
1,Restriction enzymes
2,Reverse transcriptase
3,DNA ligase
4,DNA polymerase and Polymerase Chain
Reaction
1,Restriction enzymes
In 1970,Arber,W,& H,Smith reported
microbial enzymes cut dsDNA at specific site,
These are called restriction enzymes,The discovery
indicated the first step in rDNA technology,
Restriction enzymes recognize 4 ～ 8 bases
of palindrome sequences,
Fig,7.18.(a)
Restriction enzyme
digestion of DNA
Cut to produce
Sticky ends,
or
Blunt ends,
XhoI
SciI
Fig,7.8,（ b）,（ c）
2,Reverse transcriptase
Reverse transcriptase is an RNA-
dependent DNA polymerase that uses RNA as a
template to form a DNA copy,In 1970,reverse
transcriptase is found in retroviruses to from a
DNA copy from a RNA genome template,
It synthesis single-strand cDNA from
5′→3 in the presence of a primer,a template
molecule,dNTP and Mg2+,
3,DNA ligases
DNA ligases creates a phosphodiester bond
between adjacent 3'-hydroxylated and 5'-
phosphorylated termini in duplex DNA structures,
In 1972,David Jackson et,al annealed sticky
ends of DNA fragments,followed by ligation using
DNA ligases,Thus ligation technology between
vectors and foreign DNA applied in gene cloning is
developed,and recombinant DNA molecule is
obtained,
4,DNA polymerase and polymerase
chain reaction
DNA polymerase is an enzyme that catalyzes template-
dependent synthesis of DNA from its deoxyribonucleoside 5‘-
triphosphate precursors,
In 1985,Kary Mullis et al,creates Polymerase Chain
Reaction (PCR) using DNA polymeras,It amplifies a specific
DNA fragment up to millions copies in vitro in a short period
of time,
The inventor wins Nobel Prize for the great contribution
that PCR brings to modern biotechnology,
Heat-stable DNA polymerase
from extremophiles
e.g,Taq polymerase from Thermus aquatcus
Half-life > 2 h at 95?C
Allow to perform in machine,thermal cycler
T (?C)
90
70
50
Time (min)
Cycle 1 Cycle 2 Cycle 3 Cycle
C,Genetic engineering
1,The overall strategy of genetic
engineering
2,Cloning and characterization of
target genes
3,Host and vector
4,Production of recombinant protein
1,The overall strategy of genetic
engineering (1)
A group of techniques for manipulating
DNA outside the organism from which it was
obtained,and reintroducing the recombinant or
modified DNA into another cell where it will
exert its effects,
Blue-white screening
Activity screening
E,coli expression frame
Lac promotor
RBS Ribosome binding site
ATG starting codon
MCS Multiple cloning sites
LacZ’ sequence for blue-white screening
Selective mark
E,coli replicon
Phosphorimaging
or color reaction
32P-labled probe
DIG-labled probe and marker
3,Host and vector
Cloning vector is a DNA molecule
that can replicate,and is used to transport a
piece of inserted foreign DNA into a
recipient cell,It may be a plasmid,phage,
cosmid or artificial chromosome,
----- L M Prescott et al.,in
MICROBIOLOGY,5th ed,
Vectors carry genes
into host cells
Recombination of plasmid and foreign gene
An example of a plasmid
E,coli K12 and derived strains
E,coli K12
Fe transportor
Antigen O
Toxin Capsule
? Small genome
? No plsmid
? Acapsule
? No fimbra adhesin
Fimbra
adhesin
? Antigen O reduction
? No toxin
? No Fe transportor
Wide cell
Advantages in production of recombinant
protein
a,Use of modified gene;
b,Increase of gene copies;
c,Strong promoter for transcription;
d,Optimized translation;
e,Optional regulation;
f,Easy cultivation,
4,Production of recombinant protein
a,Use of modified gene
YYYYYY
XXXXXX
YYYAAATTT CCCAAAYYY
XXXTTTAAA GGGTTTXXX
Blunting Kination
Ligation PCR
b.,c,& d,In central dogma
? lac Promoter from
E.coli
? Recombinant of lac &
trp,Ptac
? Promoter from phage
T7
? PL from E.coli phage l
? PHsh from E.coli
Induction of expression in E,coli
e,Optional regulation
PL +1 RBS FG Terminator
30℃
40 ℃
mRNA
Heat shock * ts repressor
* A temperature sensitive protein encoded by mutant cI857(ts)
Induction of gene expression in PL vector
T7 Promoter
FG
Host cell Chromosome
Vector
vector
T7 RNA
polymerase
Gene expression controlled by T7 promoter
Plac/PL
f,Easy cultivation
D,Genetic modification of organisms,gene
therapy and genetic medicines
1,Recombinant microorganisms
2,Transgenic plants
3,Transgenic animals
4,Gene therapy and genetic medicines
Definition
Metabolic (pathway) engineering
is the use of molecular techniques (or
recombinant DNA techniques) to improve
the efficiency of pathways that synthesize
specific products,
----- L M Prescott et al.,in
MICROBIOLOGY,5th ed
1,Recombinant microorganisms
Fermentation of ethanol by Zymomonas
mobilis
Entner-Doudoroff pathway
Pyruvate
Acetaldehyde + CO2
Ethanol
Pyruvate
decarboxylase
Alcohol dehydrogenase
E,coli 的代谢工程
EMP途径
乳酸 丙酮酸 乙醛
甲酸 乙醇脱氢酶
CO2+H2 乙酰辅酶 A 乙醇
磷酸转移酶 乙醛脱氢酶
乙酰磷酸 乙醛
乙酸激酶 乙醇脱氢酶
乙酸 乙醇
乳酸脱氢酶 丙酮酸脱羧酶
乙醇操纵子设计
宿主菌株的选择
基因的整合
表达水平的筛选
条件优化等
乙醇发酵的代谢工程
Production of ethanol from corn fiber hydrolysate
Strain Yield Recovery Rate
（ g/l） （ %） （ g/l /h）
Sacharomyces 1400 21.0 98 1.60
Z,mobilis CP4 22.6 88 1.04
E,coli KO11 34.7 80 1.16
EXAMPLE
Metabolic engineered strains
2,Transgenic plants
Agrobacterium spp,living in earth infects plant
cells through callus with transference of T-DNA to a
plant gnome,Thus the plant cells produce growth
regulators,and synthesize nutrition for A,spp,
A,tumefaciens cause plant cell overgrowth to
form cell tumor that forms crown gall ； A,rhizogenes
cause abnormal growth of roots cells to form,hairy
roots”,They are important tools of plant genentic
engineering,
T-DNA
intergration
Secretion
Cytopharynx
Auxin
Plant cell
Nuclear
pore
Opine
Damage
Secretion
Acetosyringon
Agrobacterium sp,
Nucleus
Pi-VirA
Induction of vir
expression
T-DNA from
Ti plasmid
VirD2 VirB
Agrobacterium tumefaciens and plants
3,Transgenic animals
Most efficient method,
Transduction of animal or animal embryo by virus,
Main purpose,
? Production of human tissue or apparatus
? Construction of animal models for human disease therapy,
? Insects,pigs,sheep,and et al,can be applied in human
protein expression,
Retrovirus
Single-strand RNA that infects mammalia;
Double-strand DNA is synthesized by RNA
reverse transcriptation after infection;
It is a ideal vector for gene therapy because
of the strong promotor,
Baculovirus
It is a insect virus of rod particles containing
double-strand DNA,which is packed up by
polyhedrin protein in vivo or expelled,
? The foreign gene can be overexpressed controlled
by the strong promoter of polyhedrin protein;
? Insect system completed the post-translation
modification;
? It is suitable for animal protein production,
4,Gene therapy and genetic medicines
Some disease is caused by gene dysfuction,
In gene therapy,a nonfunctional or dysfunctional
gene is augmented or replaced by a functional gene,
Retrovirus is always used as a vector in
gene therapy,which introduce new gene into
human multiplication cells,
Antisense RNA as genetic medicine
Some diseases are caused by abnormal
expression of a certain protein,such as most virus
disease,cancer,Alzheimer’s disease,They can be
efficiently prevented by reduction of transcription
and expression of corresponding genes,
Antisense RNA complements the sequence in
the translation initiation region of mRNA,that
efficiently stops the ribosome combination and
translation initiation,
RNA interference
RNA interference,first observed in petunias,is a
phenomenon of gene silencing at the mRNA level
offering a quick and easy way to determine the function
of a gene both in vivo and in vitro,
Duplexes of 21-23 nt RNAs (small-interfering
RNA,siRNA) produced by RNase in cytoplasm are
introduced into a cell where they associate with specific
proteins into a multi-protein siRNA complex (RNA
induced silencing complex,RISC),RISC then binds to,
and degrades the target mRNA in the cell in a highly
specific manner,
siRNA as genetic medicine
siRNA has advantages over antisense
RNA in the efficiency and specialization,
siRNA is widely applied in the clinic
therapy of eye disease and hepatitis,
It was reported in 2005 the scientists in
our country succeeded in retaining the SARS
by siRNA,
References
1,Madigan,MT,JM Martinko,and J Parker,2003,Brock Biology of
Microbiology,10th ed,Printice Hall,New Jersey,
2,Talaro,KP,2005,Foundations in Microbiology,5th ed,McGraw-Hill
co,& High Education Press,Beijing,
3,Prescott,LM,JP Harley,and DA Klein,2002,Microbiology,5th ed,
McGraw-Hill co,& High Education Press,Beijing,
4,Turner,PC,AG McLennan,AD Bates and MRH White,1997,Instant
Notes in Molecular Biolgy,Bios Scentific Publishers Limited,2000,
5,Maloy,SR,JE Cronan Jr,and D Freifelder,1994,Microbial Genetics,
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