mcb 3020, spring 2005
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MCB 3020, Spring 2005. Chapter 10: Microbial Genetics I Mutations. RNA protein. DNA. phenotype. Genetics : the study of the mechanisms of heredity and variation in organisms. DNA. Central dogma. A. The genotype determines the possible phenotypes of an organism. - PowerPoint PPT PresentationTRANSCRIPT
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MCB 3020, Spring 2005
Chapter 10:Microbial Genetics I
Mutations
2Genetics : the study of the mechanisms of heredity and variation in organisms
RNA
protein phenotype
DNADNA
Central dogma
3
Genotype the exact genetic composition(DNA sequence) of an organism
Phenotype the observable characteristics of an organism
A. The genotype determines the possible phenotypes of an organism.
4
1. Prokaryotes are relatively simple: haploid, easy to grow
3. Molecular cloning and biotechnology
B. Why study prokaryotic genetics?
2. Many principles of genetics are the same in prokaryotes and eukaryotes.
4. Control of pathogenic microorganismsTB
5
ancestral cell
extant species
mutation,DNA transfer
and natural selection(evolutionary time)
TB
Life evolves. This leads to diversity.
It is likely that all organisms are related toa single ancestral cell or group of cells.
6D. Genetic diversity can result from
1. Mutations2. DNA transfer
7Molecular Genetics I: Mutations
I. MutationsII. Effects of mutations on protein structureIII. Effects of mutations on protein functionIV. Effects of mutations on phenotypeV. Mutagens
8
inheritable changes in the genotype (DNA sequence) of an organism
I. Mutations
Mutations can play an important role in genetic diversity and evolution.
9
...GATCGGATC...
...CTAGCCTAG...
...GATAGGATC...
...CTATCCTAG...
mutation
A. A base pair change is an example of a mutation.
TB
10B. Mutations can lead to biological variation
Most mutations are harmful or neutral.
Rare beneficial mutations and naturalselection lead to new species.
TB
11C. Most mutations result from DNA replication errors.
DNA polymerases sometimes makemistakes that are not repaired.
DNA damage increases the likelihood of such mistakes.
TB
12
...GATCGGATC...
...CTAGCCTAG...DNA damage (alkylation)
...GATCGGATC...
...CTAGCCTAG...CH3 methyl-G
DNA damage can lead to mutation, but is nota mutation per se because it is not heritable.
TB
13D. Mutation frequencies are thought to be roughly similar in all organisms:
~10-9 to 10-10 / base pair / generation
Thus, in general, mutations are rare.
TB
14E. Mutants can be derived from wild-type strains (or from other mutant strains).
Wild-type: the original strain of an organism isolated from nature mutation
Mutant: an organism with a genome that carries a mutation
mutation
15Mutations in genes that encode proteins can affect
• protein structure• protein function• the phenotype of the organisms
16II. Effects of mutations on protein structure
A. base pair changes 1. silent mutations 2. missense mutations 3. nonsense mutations
B. deletionsC. insertionsD. frameshift mutationsE. inversionsF. duplications
17
Translation has two possible outcomes: (1) a change in the amino acid sequence, or (2) no change.
transcriptionmRNA
translation
OR
genemutation
Overview: Effect of mutations on protein structure
TB
18A. Base-pair changes (point mutations)
A heritable change in a single base pair of DNA
1. silent mutations2. missense mutations3. nonsense mutations
191.Silent mutations
...TAC...
...ATG...DNA
...UAU...
tyrosine
No change in the polypeptide
RNA ...UAC...
Polypeptide tyrosine
...TAT...
...ATA...mutation
TB
20
...TAC...
...ATG...DNA
...AAC...
asparagine
RNA ...UAC...
Polypeptide tyrosine
...AAC...
...TTG...mutation
2. Missense mutations
One amino acid is changed in the polypeptide. TB
21
...TAC...
...ATG...DNA
...UAG...
stop codon
RNA ...UAC...
Polypeptide tyrosine
...TAG...
...ATC...mutation
3. Nonsense mutations (mutation results in a stop codon)
A truncated polypeptide is made. TB
22
ATGAAAGAG....
ATGGAG....
B. DeletionsOne or more base pairs are lost
Possible results a. amino acids or polypeptides can be lost b. frameshifts can occur (see below) TB
23C. Insertions
ATGAAAGAG....
ATGGAG....
Possible results a. amino acids or polypeptides can be gained. b. frameshifts can occur (see below)
One or more base pairs are gained
TB
24D. Frameshift mutations
ATGCAAGTTG....one base pair deletion
Insertions or deletions that change the translational frame
Two changes in polypeptides are possible: (1) every amino acid downstream of the mutation is changed, (2) a truncated (shortened) protein is produced.
ATGAAGTTG....
TB
25DNA can have 3 reading frames:
A T G C A A G T T G A #3ala ser STOP
A T G C A A G T T G A #1met gln val
A T G C A A G T T G A #2cys lys leu
A T G C A A G T T G A
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ATGCAAGTTGA….one base pair deletion
ATCAAGTTGA
met gln val
ile lys leu
(Frameshifts occur only if insertion or deletion is in the reading frame section of a protein-encoding gene.)
Frameshift mutations change the translational reading frame.
27E. Inversionschromosomal segment is inverted
...ATGGAAGAG....
...TACCTTCTC....
...ATTTCCGAG....
...TAAAGGCTC....A number of changes in polypeptides are possible.TB
28F. Duplicationschromosomal segment is duplicated
...ATGGAAGAG....
...TACCTTCTC....
...ATGGAAGGAAGAG.... ...TACCTTCCTTCTC....
A number of changes in polypeptides are possible.TB
29III. Effects of mutations on protein function
1. No effect (common)
2. Loss of function (common)
5. Change of function (rare)
6. Restoration of function (reversion)
3. Partial loss of function (leaky)
4. Conditional loss of function
TB
301. No effect (common)
TB
Depending on the protein, up to80% of the amino acids may onlyfunction as spacers.
wild type (normal protein)
mutant protein
312. Loss of function (common)
TB
a. A change in an amino acid that participates directly in catalysis (change in the active site)
wild type (normal protein)
mutantprotein
Examples:
32
b. A change in an amino acid that causes the protein to misfold.
mutant proteinmisfoldedwild type
proteindegradation
amino acids TB
2. Loss of function (contd.)
333. Partial loss of function, "leaky" (common)
Reduction in the catalytic activityof an enzyme due to a change in 3-D shape, and / or stability.
wild type (normal)protein
mutantprotein
TB
344. Conditional loss of function (common)
e.g. Temperature-sensitive (heat- sensitive) mutations.
30°C 42°C
mutant protein
misfoldedproperlyfolded degradation
amino acids TB
355. Change of function (rare)
wild type protein
converts maltoseto 2 glucose
converts lactoseto glucose and galactose
mutantprotein
e.g. change in specificity
TB
366. Restoration of function, reversion ("back mutation") (rare)
mutantprotein nonfunctional
a second mutation
functional
TB
37a. Same site revertantsi. true revertants
A second mutation restores theoriginal DNA sequence.
ii. othersA second change at the same siteresults in a less harmful amino acidchange, or the original amino acid.
TB
38b. Second site revertants (suppressors)
i. intragenic
A second mutation at a different sitewithin the same gene restores function.
TB
ii. intergenic
A second mutation in a different gene restores function.
39
Lys 121Asp 44
a salt bridge betweenLys 121 and Asp 44 is essential to protein folding.
A mutation that converts Lys 121 to Gludestroys protein activity.A second mutation that converts Asp 44to His restores protein activity.
Note that Asp and Glu are negatively chargedand that Lys and His are positively charged. TB
Example of an intragenic suppressor
+_
40IV. Effects of mutations on phenotype
Phenotype The observable characteristics of an organism
Mutations can have manydifferent effects on phenotype.
TB
41A. Loss of enzyme activity
1. If a mutations destroys an enzyme needed for pigment formation, an albino can result.
2. If a mutation inactivates an enzyme for lactose catabolism, a microbe unable to grow on lactose will result.
TB
Examples:
42B. Loss of regulatory proteins
1. inability to induce enzymes2. inability to differentiate3. inability to tax toward nutrientsetc. etc. etc.
C. Loss of structural proteins
D. Mutations in tRNA or rRNATB
43V. Mutagens
Substances that increase mutation frequency.
In the lab, mutagens can be used to create mutations for genetic analysis.
TB
44A. Mutation frequencies
Spontaneous mutations occur with a frequency of about 10-9 / base pair / generation
TB
Mutagens are used to increase mutation frequencies to ~10-6 to 10-7 / base pair / generation(~10-3 to 10-4 / gene / generation).
45
1. Base analogs
Compounds structurally similarto the normal DNA bases
B. Types of mutagens and the mutations they cause.
TB
46
O
N
NH
O
H BrO
N
NH
O
H CH3
Thymine Bromouracil
• Bromouracil will be incorporated into DNA in place of thymine.
TB
• During DNA replication, bromouracil can mispair with guanine and cause point mutations.
472. Alkylating agentsCompounds that chemicallymodify DNA bases via alkylation
During DNA replication modified bases mispair causing single base pair change (point) mutations.
Example: dimethyl sulfateTB
483. Intercalating agentsChemicals that insert between DNA base pairs.
......
......
......
DNA basesH-bonds
backbone
Intercalating agents lead to small deletions and insertions during DNA replication.
intercalatingagent
(ethidiumbromide)
TB
494. RadiationUltraviolet light (UV)
O
N
NO
H CH3
O
N
N O
H CH3
Thymine dimer: two "T"s on the same strand become covalently bonded.
Thymine dimers lead to various replication errors.TB
501. Understand how genotype affects phenotype.2. Define mutation. Understand the role of mutations in genetic diversity and evolution. Is chemical modification of a DNA base considered a mutation? why?3. What is the most common cause of spontaneous mutations? What is the typical mutation frequency in most organisms? Define wildtype and mutant.4. What is a point mutation? Understand the effects of silent, missense and nonsense mutations on protein primary structure.5. Define deletions, insertions, frameshift mutations, inversions, and duplications. Understand how these mutations influence protein structure.6. Be able to distinguish between the different effects of mutations on protein function. What are most common effects that mutations have on protein function? Which are rare? Understand the terms leaky mutant, conditional loss of function, temperature-sensitive mutations, back mutation, reversion, revertants (know the different types), intragenic and intergenic suppression.7. Describe how a mutation might change the substrate specificity of an enzyme.8. In general how do mutations affect phenotype?9. In genetics, what is main use of mutagens? How do they affect mutation freq? Describe how base pair analogs, alkylating agents, intercalating agents and UV radiation lead to mutations. Know the examples! What is a thymine dimer?
51
MCB 3020, Spring 2005
Chapter 10:Microbial Genetics IIGenetics Techniques
52Molecular Genetics II: TechniquesI. The isolation of mutantsII. The Ames' testIII. General recombinationIV. Complementation
53Some laboratory uses of mutations
1. Mutations can help identify genes involved in particular biological processes.
2. Mutations can help to determine the function of specific genes.
(e.g. metabolic pathway genes, regulatory genes, transport genes)
54Some advantages of mutant studies with bacteria
Bacteria are haploid.
Bacteria are easily grown in large numbers.
TB
55I. The isolation of mutants
Because specific mutations are relatively rare, procedures need to be efficient. TB
To use mutations to identify genesand their functions, the first step is to create and isolate organisms with mutations that affect the process of interest (e.g. histidine biosynthesis).
56
(strains with mutations in his biosynthetic genes)
1. Designate a particular E. coli strain as the wildtype strain (His+ phenotype).
TB
A. Isolation of histidine biosynthetic mutants in Escherichia coli
2. Grow a broth culture of the wildtype.
57
dilute and plateon a rich medium
TB
mutagenized culture
3. Treat culture with mutagen (produces mutations at random locations).
58
Replica plating
minimal mediumwithout histidine
minimal mediumwith histidine
4. Select or screen for mutant strains that require histidine for growth (His- phenotype).
59Replica plating
minimalmedium
minimalmedium + histidine
Simultaneous transfer of all colonies on master plate to several different media.
Between 1/1000 and 1/10,000 colonies will havea mutation in a particular gene. TB
605. Prepare pure cultures from strains that require histidine for growth.
7. Write down a list of mutant strains that indicates their genotype and phenotype.
6. These strains have mutations in genes needed for histidine biosynthesis.
TB
61In practice, the genotype and phenotype of the mutant strains is indicated as theirdifferences from the wildtype strain.
Strain # genotype12
his-1his-2
phenotypeHis-His-
Eventually the dash replaced by a letter designating a specific gene
TB
62
Mutant genes hisC1, hisC2, hisC3
Naming genes and mutations (Example for a histidine biosynthetic gene)
Gene hisC
Protein HisC or (name of protein)
Phenotypes His+ (can make histidine) His- (cannot make histidine)
63AuxotrophsA His- mutant of E. coli cannot make histidine and requires histidine as a growth factor
•
An E. coli mutant with a His- phenotype is a histidine auxotroph.
•
It will grow in the presence, but not in the absence, of histidine.
•
Auxotroph: a nutritional mutant that hasa requirement for a growth factor (relative to its parent strain, the prototroph)
•
64B. How could you isolate mutants in lactose catabolism?
Use the same procedure as above,but screen for mutant strains unable to catabolize lactose.
TB
A convenient screen for lactose catabolism is the MacConkey indicator medium.
65MacConkey lactose indicator medium
Detects acids produced from lactose catabolism.
Lac+
Lac- lac mutants are unable to produce acid and hence are white.
Between 1/1000 and 1/10,000 colonies will havea mutation in a particular gene. TB
66
1. Screening Identification of particular mutants by comparing their properties to the wild type (usually colony properties).
Examples indicator plates (see above)loss of pigmentation
C. Screening and Selection
TB
672. SelectionIdentification of particular mutants by using conditions that prevent the growth of other cells.
TB
e.g. selection for antibiotic resistance
medium with antibiotic (plate ~108 cells)
Colonies that grow are antibiotic-resistantmutants
68
1. Spread ~108 His- cells on minimal plates (no histidine)
2. Soak filter disk with test compound and place on plate.
3. Incubate plates and examine (look for increase in # of back mutations that restore His+ phenotype)
II. The Ames test A test used to identify mutagens
TB
minimal
69Possible resultstest compound
#1test compound
#2
is not amutagen
is a mutagen
TB
revertants
control
70
TB
III. General recombination
DNA rearrangements involving crossovers between homologous DNA sequences.
71A. Cellular uses of recombination
1. The generation of genetic variation in eukaryotes during meiosis
3. DNA Repair
2. The generation of variation in prokaryotes via its role in gene transfer
TB
72B. Genetic crossovers
breakage of phosphodiester bonds
x "x" is the crossover site
DNA exchanges used in recombination
TB
73
reunion of phosphodiester bonds
For general recombination, crossovers only occur between between homologous (identical or nearly identical) DNA segments.
TB
74C. Outcomes of recombination
X
A B C
A B Csingle crossover
1. identicalsequences
A B C
A B C
identicalsequences
TB
75
X
A B C
a B c
a B C
A B c
single crossover
2. nearlyidenticalsequences
recombinantsequences
TB
763. Two circular DNA molecules
Xsingle crossover
integration
TB
774. circular + linear
X
double crossover
X
TB
785. single strand exchange
TB
79D. A model for recombination
alignment ofhomologoussequences
nicking
TB
80
unwinding (RecBCD)
strand invasion (RecA)
TB
81
strand exchange
From this point there are twomain methods of resolution.
TB
821. Inner strand resolution (break and religate inner strands)
inner
break
TB
83
religate
TB
842. Outer strand resolution (break and religate outer strands)
outer strandsbreak
TB
85
religate
a
a
bb
a ab b
cd
dc
cd
cd
TB
86IV. Complementation
In a cell that has a recessive mutation, restoration of the wild type phenotype by a second DNA molecule.
TB
AA
87A. In laboratory research, complementation has important uses.
1. Screening for clones of interest.
2. Verification that a particular mutation results in a particular phenotype
TB
88
chromosome
A
Mutation 1
1. Start with a bacterium with a recessive mutation resulting in Trp– phenotype or any other phenotype.
B. How complementation is observed
TB
892. Introduce a second DNA molecule into the bacterium by one of several methods.
mutation 1
A
Restoration of the wildtype (in this case Trp+)phenotype is termed complementation. TB
second DNA molecule (plasmid)
A
90
Complementation indicates that the second DNA molecule (the plasmid) has a good copy of the chromosomal gene that is mutated.
C. Key Point
TB
91Study objectives1. Describe two ways that mutations are used in laboratory research. 2. Describe how bacterial biosynthetic and catabolic mutants can be isolated. What is replica plating?3. Know how to write the names of genes, mutant genes, protein products, and phenotypes. Pay particular attention to font and capitalization. 4. What are prototrophs and auxotrophs?5. Compare and contrast screens and selections. Are screens and selections equally useful for the isolation of very rare mutations?6. Understand what the Ames test is used for and how it works.7. Name three cellular uses of general recombination.8. What are the constraints of genetic crossovers in general recombination?9. Describe recombination, its main steps, and the key enzymes involved. I will NOT ask about inner strand versus outer strand resolution.10. What is complementation and what are its uses? What is the role of plasmids in complementation?
92
MCB 3020, Spring 2005
Chapter 10: Microbial Genetics III:
DNA transfer
93
ancestral cell
extant species
mutation,DNA transfer
and natural selection(evolutionary time)
TB
Life evolves. This leads to diversity.
It is likely that all organisms are related toa single ancestral cell or group of cells.
94Molecular Genetics III: DNA TransferI. DNA transfer in prokaryotes
A. transformationB. transductionC. conjugation
II. Transposable elements
95I. DNA transfer in prokaryotes
A. transformationB. transductionC. conjugation
TB
96DNA transfer in prokaryotes
The transfer of donor DNA tothe genome of a recipient cell.
TB
97Uses of DNA transfer1. In natural environments, DNA
transfer is used to generategenetic variation.
2. In the lab, DNA transfer is used forgenetic mapping and the
construction of recombinant organisms with particular genotypes.
TB
98A. Transformation
Transfer of free DNA to a bacterial genome.
TB
free DNA
99free DNA
recipient cell
general recombination
transformant
chromosome
TB
100
General recombination is not necessary because plasmids have origins of replication
transformantfree DNA (plasmid)
TB
1011. CompetenceThe capacity of cells to take up free DNA.
a. Some cells are naturally competent
b. In some cells, competence can be induced by chemical and physical treatment
c. In most cells, competence can be induced by electroporation.
TB
102Electroporation
+ electrode- electrode
cells + DNA
A brief electric pulse induces poresin the cell envelope and free DNA enters
TB
103B. Transduction
Transfer of DNA by viral particles
1. Generalized transduction2. Specialized transduction
TB
1041. Generalized transduction
In generalized transduction, transducing particles formed by packaging errors can contain DNA from any part of the donor genome.
105
donor cell
many viruses
viral replication
chromosome
a few transducing particles containing part ofdonor cell DNA (formed by packaging errors)
Generalized transduction
TB
106
recipient cell
transducing particle
general recombination
transductant
recipient chromosome
TB
107
prophage(integrated bacterial virus)improper
excision
donor cell (lysogen)
transducingparticles
viralreplication
2. Specialized transduction
chromosome
TB
108recipient cell transducing
virus
transductants
phageintegration
chromosome
generalrecombination
TB
109Key points for specialized transductionTransducing viruses formed by improper excision can only transfer DNA adjacent to the prophage insertion site.
Transducing viruses can become part of the recipient genome by general recombination or integration.
TB
110C. Conjugation
Direct cell to cell DNA transfer involving certain plasmids.
TB
Picture7
1111. Conjugative plasmidsplasmids that mediate their own transfer
e.g. F-plasmid
oriS
oriT
tra ISIS
TB
112
F plasmid
donor cell (F+)
F- pilus
2. The DNA transfer process
The F-pilus is used for cell-cell attachment
recipient cell (F-)
chromosomechromosome
TB
113
replication andtransfer of ssDNA
F+
exconjugant
F+
Note that the recipient cell becomes F+ TB
1143. The F-plasmid can integrate into the bacterial chromosome.
chromosome
Hfr strain integratedF plasmid
Integrated F plasmids can transfer the chromosome.
F plasmid
integration
TB
115
A cell that carries the integratedF plasmid is called the "Hfr" strain
Hfr stands for "high frequency of recombination"
a. Hfr strain
116
A bacterium with an F plasmid integrated into its chromosome.
Hfr strain
In the lab, they are used for genetic mapping (determining gene location).
In nature, they can transfer genes and play a role in generating variation.
integratedF plasmidHfr strain
TB
117b. The integrated F plasmid can mobilize the chromosome (i.e. move part of the chromosome to another cell)
integratedF plasmid
Hfr strain
TB
118Bacterial genetic mapsmaps that show the relative locationsof genes.
hisGDCBHAFIE
galKTEspoT
~500-12,000 genes TB
X
119c. The integrated F-plasmid can excise improperly, forming a plasmid with part of the host chromosome. The resulting plasmid is called F-prime (F').
Hfr strainintegratedF plasmid
F'
Improper excision
TB
120F-prime (F'):
Hfr strain
F plasmid
F'
improperexcision
An improperly excised F plasmid containing a segment of bacterial chromosomal DNA.
TB
121F-prime (F')
part of F plasmid
segment of bacterialchromosome
Formed by improper excision of HfrCan transfer chromosomal genes TB
122III. Transposable elements
A. examples1. insertion sequences2. transposons
B. transpositionC. uses of transposable elements
TB
123Transposable elementsDNA segments that can move from onelocation to another; "mobile DNA"
transposable element
IS IS
host DNA
disrupted, nonfunctional gene TB
124
Their function is uncertain.They may simply be "selfish DNA".
In the lab, transposons are used to createmutations.
Found as part of the genome of all organisms carefully examined
IS IS transposable element
TB
125
1. Insertion sequences
IS2 tnp
inverted terminal repeat
tnp = transposase genetransposase catalyzes transposition
A. Examples of transposable elements
TB
1262. Transposons
kan str ble
IS50L IS50R
tnpTn5
host genes
Transposons typically consist of host gene(s)flanked by insertion sequences. TB
127B. TranspositionThe movement of a transposable element
1. conservative
One element is moved to another site.
TB
128Transposition
2. replicative
One element is duplicated.The first copy stays at the original site.The second copy is found at another site.
TB
129C. Uses of transposable elements
1. In the lab, transposon mutagenesis
2. In nature, function is not known.Just selfish DNA?
TB
130Study objectives1. Know that DNA transfer can generate genetic variation and can be used to construct recombinant organisms. 2. Know how competence affects transformation and how competence is induced. 3. Know how DNA is transferred by transformation, transduction, and conjugation. Be able to compare and contrast these types of gene transfer.4. Know what Hfr strains and F-prime plasmids are.5. Be able to compare and contrast transposons and insertion sequences. 6. Understand conservative and replicative transposition and know that transposition causes mutations.