Chapte8 Microbial genetics
8.1 DNA as genetic material
8.2 Mutation
8.3 DNA repair
Microbial molecular biology and
genetics
? A clone,a population of cells that are
derived asexually from a parental cell and
are genetically identical.
? Genome,all the genes present in a cell or
virus.
? Central dogma,DNA?RNA ? protein
transcription translation
8.1 DNA as genetic material
The Hershey-Chase experiments
RNA as genetic material
? TMV
8.2 Mutations and Mutants
? Mutations,initially characterized as altered phenotypes or
phenotypic expression,a mutation is a stable,heritable
change in the nucleotide sequence of DNA.
? Phenotype,the observable characteristics of an organism
– designated by a capital letter followed by two small
letters,with either a plus and minus superscript to
indicate the presence or absence of that property,His-,
Glu+ et al.
? Genotype,The precise genetic make-up of an organism
– designated by three lower case letters followed by a
capital letter (all in italics) indicating the particular gene
involved,hisC,mutations in the hisC gene would be
designated as hisC1,hisC2 et al.
Types of mutations
? Conditional mutations,those that are expressed only
under certain environmental conditions.
eg,E.coli,a conditional lethal mutant,low temperature
grow; high temperature die.
? Biochemical mutations,those causing a change in the
biochemistry of the cell,Mutations make a
microorganism unable to grow on a medium lacking an
adequate supply of the pathway’s end product.
auxotrophs,the mutant cannot grow on minimal
medium (MM) and requires nutrinent supplements.
prototrophs,microbial strains that can grow on MM.
? Resistant mutant,a particular type of biochemical
mutant that acquires resistance to some pathogen,
chemical,or antibiotic.
Mutations occur in one of two
ways:
? 1,Spontaneous mutations,arise
occasionally in all cells and develop in the
absence of any added agent.
? 2,Induced mutations,the result of exposure
of the organism to some physical or
chemical agent called a mutagen.
Spontaneous mutations
without exposure to external agents
? 1,Errors in DNA replication
? 2,The action of transposons
? 3,Frameshifts by the deletion or addition of
bases
? 4,The insertion sequences of DNA
segments into genes
? Transition mutations,purine for
putine or pyrimidine for pyrimidine
substitutions.
relatively common
? Transversion mutations,a purine is
substituted for a pyrimidine,or a
pyrimidine for a purine.
rarer
Apurinic site,purine nucleotides lose their
base.
Apyrimidinic site,cytosine can be
deaminated to uracil,which is removed.
Reactive forms of oxygen,O·,peroxides
eg,
C-G ?8-oxo-7,8-dihydrodeoxyguanine-A
Induced mutations,mutagens
? 1,Base analogs,eg,5-BU,an analogs of T.
keto and enol,keto-A; enol-G
? 2,Specific mispairing,a mutagen changes a base’s structure
and therefore alters its base pairing characteristics.
eg,methyl-nitrosoguanidine,an alkylating agent that adds
methyl groups to guanine,causing it to mispair with
thymine,GC-AT transition.
methyl-C-T
? 3,Intercalating agent,distort DNA to induce single
nucleotide pair insertions and deletions.
eg,proflavin and acridine orange
? 4,Damaged DNA can no longer act as a template.
uv radiation; carcinogens,aflatoxins B1; benzopyrene
derivatives.
Mutagens
Chemical mutagens:
Base analogs
Chemicals reacting
with DNA
Alkylating agents
Physical mutagens:
UV
Ionizing rdiation
Physical mutagen,Radiation
? Noninonizing (electromagnetic):
– UV,induction of pyrimidine dimers
? Ionizing radiation
– X-ray,cosmic rays,gamma rays; causing water
and other substances to ionize; such as the
generation of hydroxyl radical which reacts with
DNA
Molecular
basis of
Mutation
? Point Mutation(one
base pair mutant)
– Silent mutation
– Nonsense mutation
– Missense
mutation(the effect
may range from
complete loss of
activity to no change
at all)
天
冬
酰
胺
酪
氨
酸
Frameshift mutations
? Occur in protein-encoding gene that including
the reading frame
? If occurs in promoter region,it is not frameshift
Back Mutations or Reversions
? Point Mutations are reversible
? Revertant
– wild type phenotype that was lost in the
mutant is restored.
? Same site revertants
? True revertants
? Second site mutation
–Suppressor mutation,restore the wild type
phenotype by mutation somewhere else.
Rates of Mutation
? Spontaneous mutation,10-6,
? Transposition,10-4
? Nonsense mutation,10-6-10-8
? Missense mutation,10-6-10-8
Hypermutation
Some starving bacteria might rapidly
generate multiple mutations through activation
of special mutator genes,This would produce
many mutant bacterial cells,In such a random
process,the rate of production of favorable
mutants would increase,with many of these
mutants surviving to be counted,There would
appear to be directed or adaptive mutation
because many of the unfavorable mutants
would die.
Isolation of Mutants
? Pigmented and
nonpigmented
mutants
? Antibiotic-resistant
mutants within the
inhibition zone
? Genetic marker,
such as the
expression of b-
galactosidase that
produces the blue
color
Replica Plating Technique,Isolation of Mutants
Velveteen (锦绒布）
Auxotroph (营养缺陷型 )
Prototroph (原养型 )
Replica Plating Technique,Isolation of Mutants
? Penicillin-Selection Method
– Penicillin kills only growing cells,If penicillin is added to a
population growing in a medium lacking the growth factor
required by the designed mutant,the parent cells will be killed,
whereas the non-growing mutant cells will be unaffected.
Mutants that are detectable
? Non-motile
? Non-capsulated
? Rough colony
? Nutritional
? Sugar fermentation
? Drug resistant
? Virus resistant
? Temperature sensitive
? Pigmentless
? Cold sensitive
Mutagenesis and Carcinogenesis
The Ames Test
? Bacteria can be used as screening agents for
the potential mutagenicity of chemicals
– As it has been found that many mutagenic chemicals are
also carcinogenic,capable of causing cancer in animals and
humans
? The fact that a compound is mutagenic in a bacterial
system serves as a warning of possible danger
? Dr,Bruce Ames at the UC Berkeley has developed
the Ames Test technique for the screening of
carcinogenic compounds.
The Ames Test
? Histidine auxotrophs of
Salmonella typhimurium
and tryptophan auxotrophs
of Escherichia coli have
been the major tools for the
Ames test.
? Induction of a phage lambda
lysogen has also been used
as an assay of DNA damage
Other Methods of Creating Mutations
? Biological mutagens
– Transposon mutagenesis,bacteriophage Mu
? Site-directed mutagenesis
– Technique has been developed to make
mutation in a specific DNA site,
8.3 DNA repair
? 1,Excision repair,first excise,then DNA
polymerase I,at last DNA ligase join
? 2,Removal of lesions (photoreactivation)
splitting thymine dimers apart into separate
thymines with the help of visible light.
? 3,Postreplication repair
mismatch repair system,excise ? replace ? seal
? 4,Recombination repair,recA protein
? 5,SOS repair
SOS regulatory system
Mutations may arise from error prone DNA repair
Questions
? Experimental evidence of DNA as genetic
material?
? Types of mutations
? What is Mutagens?
? Molecular basis of Mutation
? Rates of Mutation
? How to detect and isolate mutant?
? The Ames Test
? What are DNA repair?
8.1 DNA as genetic material
8.2 Mutation
8.3 DNA repair
Microbial molecular biology and
genetics
? A clone,a population of cells that are
derived asexually from a parental cell and
are genetically identical.
? Genome,all the genes present in a cell or
virus.
? Central dogma,DNA?RNA ? protein
transcription translation
8.1 DNA as genetic material
The Hershey-Chase experiments
RNA as genetic material
? TMV
8.2 Mutations and Mutants
? Mutations,initially characterized as altered phenotypes or
phenotypic expression,a mutation is a stable,heritable
change in the nucleotide sequence of DNA.
? Phenotype,the observable characteristics of an organism
– designated by a capital letter followed by two small
letters,with either a plus and minus superscript to
indicate the presence or absence of that property,His-,
Glu+ et al.
? Genotype,The precise genetic make-up of an organism
– designated by three lower case letters followed by a
capital letter (all in italics) indicating the particular gene
involved,hisC,mutations in the hisC gene would be
designated as hisC1,hisC2 et al.
Types of mutations
? Conditional mutations,those that are expressed only
under certain environmental conditions.
eg,E.coli,a conditional lethal mutant,low temperature
grow; high temperature die.
? Biochemical mutations,those causing a change in the
biochemistry of the cell,Mutations make a
microorganism unable to grow on a medium lacking an
adequate supply of the pathway’s end product.
auxotrophs,the mutant cannot grow on minimal
medium (MM) and requires nutrinent supplements.
prototrophs,microbial strains that can grow on MM.
? Resistant mutant,a particular type of biochemical
mutant that acquires resistance to some pathogen,
chemical,or antibiotic.
Mutations occur in one of two
ways:
? 1,Spontaneous mutations,arise
occasionally in all cells and develop in the
absence of any added agent.
? 2,Induced mutations,the result of exposure
of the organism to some physical or
chemical agent called a mutagen.
Spontaneous mutations
without exposure to external agents
? 1,Errors in DNA replication
? 2,The action of transposons
? 3,Frameshifts by the deletion or addition of
bases
? 4,The insertion sequences of DNA
segments into genes
? Transition mutations,purine for
putine or pyrimidine for pyrimidine
substitutions.
relatively common
? Transversion mutations,a purine is
substituted for a pyrimidine,or a
pyrimidine for a purine.
rarer
Apurinic site,purine nucleotides lose their
base.
Apyrimidinic site,cytosine can be
deaminated to uracil,which is removed.
Reactive forms of oxygen,O·,peroxides
eg,
C-G ?8-oxo-7,8-dihydrodeoxyguanine-A
Induced mutations,mutagens
? 1,Base analogs,eg,5-BU,an analogs of T.
keto and enol,keto-A; enol-G
? 2,Specific mispairing,a mutagen changes a base’s structure
and therefore alters its base pairing characteristics.
eg,methyl-nitrosoguanidine,an alkylating agent that adds
methyl groups to guanine,causing it to mispair with
thymine,GC-AT transition.
methyl-C-T
? 3,Intercalating agent,distort DNA to induce single
nucleotide pair insertions and deletions.
eg,proflavin and acridine orange
? 4,Damaged DNA can no longer act as a template.
uv radiation; carcinogens,aflatoxins B1; benzopyrene
derivatives.
Mutagens
Chemical mutagens:
Base analogs
Chemicals reacting
with DNA
Alkylating agents
Physical mutagens:
UV
Ionizing rdiation
Physical mutagen,Radiation
? Noninonizing (electromagnetic):
– UV,induction of pyrimidine dimers
? Ionizing radiation
– X-ray,cosmic rays,gamma rays; causing water
and other substances to ionize; such as the
generation of hydroxyl radical which reacts with
DNA
Molecular
basis of
Mutation
? Point Mutation(one
base pair mutant)
– Silent mutation
– Nonsense mutation
– Missense
mutation(the effect
may range from
complete loss of
activity to no change
at all)
天
冬
酰
胺
酪
氨
酸
Frameshift mutations
? Occur in protein-encoding gene that including
the reading frame
? If occurs in promoter region,it is not frameshift
Back Mutations or Reversions
? Point Mutations are reversible
? Revertant
– wild type phenotype that was lost in the
mutant is restored.
? Same site revertants
? True revertants
? Second site mutation
–Suppressor mutation,restore the wild type
phenotype by mutation somewhere else.
Rates of Mutation
? Spontaneous mutation,10-6,
? Transposition,10-4
? Nonsense mutation,10-6-10-8
? Missense mutation,10-6-10-8
Hypermutation
Some starving bacteria might rapidly
generate multiple mutations through activation
of special mutator genes,This would produce
many mutant bacterial cells,In such a random
process,the rate of production of favorable
mutants would increase,with many of these
mutants surviving to be counted,There would
appear to be directed or adaptive mutation
because many of the unfavorable mutants
would die.
Isolation of Mutants
? Pigmented and
nonpigmented
mutants
? Antibiotic-resistant
mutants within the
inhibition zone
? Genetic marker,
such as the
expression of b-
galactosidase that
produces the blue
color
Replica Plating Technique,Isolation of Mutants
Velveteen (锦绒布）
Auxotroph (营养缺陷型 )
Prototroph (原养型 )
Replica Plating Technique,Isolation of Mutants
? Penicillin-Selection Method
– Penicillin kills only growing cells,If penicillin is added to a
population growing in a medium lacking the growth factor
required by the designed mutant,the parent cells will be killed,
whereas the non-growing mutant cells will be unaffected.
Mutants that are detectable
? Non-motile
? Non-capsulated
? Rough colony
? Nutritional
? Sugar fermentation
? Drug resistant
? Virus resistant
? Temperature sensitive
? Pigmentless
? Cold sensitive
Mutagenesis and Carcinogenesis
The Ames Test
? Bacteria can be used as screening agents for
the potential mutagenicity of chemicals
– As it has been found that many mutagenic chemicals are
also carcinogenic,capable of causing cancer in animals and
humans
? The fact that a compound is mutagenic in a bacterial
system serves as a warning of possible danger
? Dr,Bruce Ames at the UC Berkeley has developed
the Ames Test technique for the screening of
carcinogenic compounds.
The Ames Test
? Histidine auxotrophs of
Salmonella typhimurium
and tryptophan auxotrophs
of Escherichia coli have
been the major tools for the
Ames test.
? Induction of a phage lambda
lysogen has also been used
as an assay of DNA damage
Other Methods of Creating Mutations
? Biological mutagens
– Transposon mutagenesis,bacteriophage Mu
? Site-directed mutagenesis
– Technique has been developed to make
mutation in a specific DNA site,
8.3 DNA repair
? 1,Excision repair,first excise,then DNA
polymerase I,at last DNA ligase join
? 2,Removal of lesions (photoreactivation)
splitting thymine dimers apart into separate
thymines with the help of visible light.
? 3,Postreplication repair
mismatch repair system,excise ? replace ? seal
? 4,Recombination repair,recA protein
? 5,SOS repair
SOS regulatory system
Mutations may arise from error prone DNA repair
Questions
? Experimental evidence of DNA as genetic
material?
? Types of mutations
? What is Mutagens?
? Molecular basis of Mutation
? Rates of Mutation
? How to detect and isolate mutant?
? The Ames Test
? What are DNA repair?
