Transcript Ch8MicrobialGenetics

Objectives
Define genetics, genome, chromosome, gene, genetic code,
genotype, phenotype, and genomics.
Describe the process of DNA replication.
Describe protein synthesis, including transcription, RNA
processing, and translation.
Classify mutations by type, and describe how mutations are
prevented and repaired.
Define mutagen.
Describe two ways mutations can be repaired.
Outline methods of direct and indirect selection of mutants.
Identify the purpose and outline the procedure for the Ames
test.
Compare the mechanisms of genetic recombination in
bacteria.
Differentiate between horizontal and vertical gene transfer.
Describe the functions of plasmids and transposons.
© 2004 by Jones and Bartlett Publishers
Terminology
Complementary but antiparallel
 Genetics
 Genome
 Gene
 Chromosome
 Base pairs
 Genetic code
 Genomics
 Genotype
 Phenotype
DNA
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The Bacterial DNA
 Mostly single circular chromosome
 Attached to plasma membrane
 DNA is supercoiled
 Number of genes
in E. coli
 Extra-chromosomal
bacterial DNA:
_________(1-5% of chromosome size)
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E. coli
Fig 8.1
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Figure 8.1a
Chromosome Map of E. coli
Chromosome length:
 1mm
Cell length ?
Figure 8.1b
Flow of Genetic Information
Fig 8.2 – Foundation Figure
DNA Replication
 DNA polymerase
initiated by RNA primer
 bidirectional
 origin of replication
 leading strand:
continuous DNA
synthesis
 lagging strand:
discontinuous DNA
synthesis  Okazaki
fragments
 semiconservative
2
Replication fork
Replication in 5'  3' direction
Fig 5.8
Replication 1; 2; 3
of circular
bacterial
Chromosome
Fig 8.6
Protein Synthesis
Genetic code: universal and
degenerate (or redundant)
Fig 8.8
Transcription
 produces 3 types of RNA (?)
 Enzyme necessary ?
 Promoters and terminators
Fig 8.7
Translation
 produces the protein
 Sense codons vs. nonsense codons
 anticodons
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Fig 8.9
Compare to Fig 8.8
Transcription
 RNA polymerase binds to promotor sequence
 proceeds
in 5'  3'
direction
 stops when
it reaches
terminator
sequence
Fig 8.7
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More Details on Translation
 Nucleotide sequence of mRNA is translated into
amino acid sequence of protein using “three
letter words” = codons
 Translation of mRNA begins at the start codon:
AUG
 Translation ends at a stop codon: UAA, UAG,
UGA
 Requires various accessory molecules and 3
major components: ?
 In Prokaryotes: Simultaneous transcription and
translation  Polyribosomes
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The Translation Process in Protein
Synthesis
Compare to Fig 8.9
Continuous Transcription
and Translation
Simultaneous
Transcription
and Translation
in Prokaryotes
Compare to Fig 8.10
Mutations
Change in genetic material.
1. Point mutations = base pair substitution (silent,
missense, nonsense)
2. Frameshift mutations = Insertion or deletion of one
or more nucleotide pairs
Review
Fig 8.17
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Various Point Mutations
Missense
Nonsense
Silent
Fig 8.17
Mutations cont.
 May be neutral (silent), beneficial, or harmful.
 Spontaneous mutation rate  10-6  1 mutation
per million replicated genes
 Mutagens increase mutation rate 10 – 1000x
Chemical mutagens
 Nucleoside (base) analogs have altered basepairing properties. They can be


randomly incorporated into growing cells (cancer drugs)
only used by viral enzymes (e.g. AZT)
 Frameshift mutagens such as intercalating
agents (e.g.:, aflatoxin, ethidium bromide)
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Fig 8.19a
Distortion due to
intercalating agent
will lead to one or
more base-pairs
inserted or deleted
during replication.
Potent carcinogens!
Radiation as a Mutagen
1. Ionizing radiation (x-rays and -rays)
lead to deletion mutations (ds breaks)
2. UV rays lead to thymine
dimers (intrastrand bonding)
 Photolyases = light repair enzymes
(use energy from visible light to fix UV light
damage)
 Nucleotide excision repair for repair of
all mutations
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Repair
 Photolyases separate thymine dimers
 Nucleotide excision repair
Fig 8.20
ANIMATION Mutations: Repair
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Mutagen Identification: Ames
Test
 Wild type vs. mutant
 Auxotroph vs. prototroph
 Many mutagens are carcinogens
Combine animal liver cell extracts with Salmonella
auxotroph
 Expose mixture to test substance
 Examine for signs of mutation in Salmonella, i.e.
Look for cells (colonies) that have reverted from his– to
his+
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Ames Reverse Gene Mutation Test
Fig. 8.22
Professor Richard A. Muller of
UC Berkeley on the Ames Test
and Natural Foods
Positive or
negative
Ames
test?
Explain
what
happened
Genetic Transfer and Recombination
 Vertical gene transfer: Occurs during
reproduction between generations of cells.
 Horizontal (lateral) gene transfer: Transfer
of genes between cells of the same
generation. Leads to genetic recombination
 Three mechanisms of horizontal gene
transfer:
 Transformation
 Conjugation
 Transduction
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Genetic Recombination
Vertical gene transfer: Occurs
during reproduction between
generations of cells.
Horizontal gene transfer: The
transfer of genes between cells of
the same generation. Leads to
genetic recombination.
Three mechanisms of horizontal
gene transfer:
1. Transformation
2. Conjugation
3. Transduction
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as Benjamin Cummings
ANIMATION
Horizontal Gene Transfer: Overview
Genetic Recombination
 Exchange of
genes between
two DNA
molecules
 Crossing over
occurs when two
chromosomes
break and rejoin
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Figure 8.23
1) Transformation
“Naked” DNA transfer
Recipient cells have to be “competent”
Occurs naturally among very few genera (G+
and G–)
Simple laboratory treatment will make E. coli
competent  workhorse for genetic
engineering
Griffith’s historical experiment in 1928
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Griffith’s Experiment to Demonstrate Genetic
Transformation
Fig 8.24
ANIMATION Transformation
Transformation
and
Recombination
Fig 8.25
2) Conjugation
Plasmid and chromosomal DNA
transfer via direct cell to cell contact
High efficiency
F+ = donor cell. Contains F plasmid
(factor) and produces conjugation
(F) pilus (aka “sex pilus”)
Recipient cell (F– ) becomes F+
Fig 8.26
In some cells F factor integrates into
chromosome  Hfr cell
R plasmids (R factors) are also transferred via
conjugation
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ANIMATIONs
Fig 8.27
3) Transduction
DNA Transfer from donor to
recipient cell with help of
bacteriophage (= transducing
phage)
2 types of phage-bacteria interaction:
1. Generalized transduction happens via
lytic cycle caused by virulent phages
Fig 8.27
2. Specialized transduction will be covered in
Ch 13
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Transduction by a
Bacteriophage
ANIMATION Generalized
Transduction
ANIMATION Specialized
Transduction
Fig 8.28