Download Chapter 8 Bacterial Genetics

Survey
yes no Was this document useful for you?
   Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Document related concepts

Polycomb Group Proteins and Cancer wikipedia, lookup

Transposable element wikipedia, lookup

Human genome wikipedia, lookup

NEDD9 wikipedia, lookup

Gel electrophoresis of nucleic acids wikipedia, lookup

Nucleosome wikipedia, lookup

Epigenetics of human development wikipedia, lookup

United Kingdom National DNA Database wikipedia, lookup

Minimal genome wikipedia, lookup

Epistasis wikipedia, lookup

Genealogical DNA test wikipedia, lookup

Mitochondrial DNA wikipedia, lookup

DNA polymerase wikipedia, lookup

Nutriepigenomics wikipedia, lookup

Zinc finger nuclease wikipedia, lookup

Plasmid wikipedia, lookup

Primary transcript wikipedia, lookup

DNA repair wikipedia, lookup

Epigenomics wikipedia, lookup

Genomics wikipedia, lookup

Genome (book) wikipedia, lookup

Genomic library wikipedia, lookup

Nucleic acid double helix wikipedia, lookup

DNA vaccination wikipedia, lookup

Replisome wikipedia, lookup

Genome evolution wikipedia, lookup

Molecular cloning wikipedia, lookup

DNA supercoil wikipedia, lookup

Cell-free fetal DNA wikipedia, lookup

Non-coding DNA wikipedia, lookup

Genetic engineering wikipedia, lookup

Gene wikipedia, lookup

Microsatellite wikipedia, lookup

DNA damage theory of aging wikipedia, lookup

Cancer epigenetics wikipedia, lookup

Designer baby wikipedia, lookup

Oncogenomics wikipedia, lookup

Therapeutic gene modulation wikipedia, lookup

Frameshift mutation wikipedia, lookup

Mutagen wikipedia, lookup

Extrachromosomal DNA wikipedia, lookup

Cre-Lox recombination wikipedia, lookup

Vectors in gene therapy wikipedia, lookup

Deoxyribozyme wikipedia, lookup

Nucleic acid analogue wikipedia, lookup

Genome editing wikipedia, lookup

Site-specific recombinase technology wikipedia, lookup

Mutation wikipedia, lookup

No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia, lookup

Helitron (biology) wikipedia, lookup

Artificial gene synthesis wikipedia, lookup

History of genetic engineering wikipedia, lookup

Point mutation wikipedia, lookup

Microevolution wikipedia, lookup

Transcript
Chapter 08
Lecture Outline
See separate PowerPoint slides for all figures and tables preinserted into PowerPoint without notes.
Copyright © McGraw-Hill Education. Permission required for reproduction or display.
1
A Glimpse of History
 Barbara McClintock (1902–1992)
• Observed that kernel colors in corn not inherited in a
predictable manner
• Concluded segments of DNA moved in and out of genes
involved in color
• Destroyed function of genes
• Published results in 1950
• Met with skepticism
• DNA believed stable
• Idea established by 1970s
• Awarded Nobel Prize in 1983
• Termed transposons
• “Jumping genes”
Antibiotic Resistance
 Staphylococcus aureus
• Gram-positive coccus; commonly called Staph
• Frequent cause of skin and wound infections
• Since 1970s, treated with penicillin-like antibiotics
• For example, methicillin
• In 2004, over 60% of S. aureus strains from
hospitalized patients were resistant to methicillin
• ~2.3 million healthy people in U.S. harbor methicillinresistant S. aureus (MRSA)
• Healthcare-associated MRSA (HA-MRSA) resistant to
other antibiotics, including vancomycin
• Vancomycin considered drug of last resort
8.1. Genetic Change in Bacteria
 Organisms adapt to changing environments
• Natural selection favors those with greater fitness
• Bacteria adjust to new circumstances
• Regulation of gene expression (Chapter 7)
• Genetic change (Chapter 8)
• Bacteria excellent system for genetic studies
• Rapid growth, large numbers
• More known about E. coli genetics than any other
• Change in organism’s DNA alters genotype
• Sequence of nucleotides in DNA
• Bacteria are haploid, so only one copy, no backup
• May change observable characteristics, or phenotype
• Also influenced by environmental conditions
8.1. Genetic Change in Bacteria
 Mutation and horizontal gene transfer
8.1. Genetic Change in Bacteria
 Mutations can change organism’s phenotype
• Deletion of gene for tryptophan biosynthesis yields
mutant that only grows if tryptophan supplied
• Growth factor required; mutant termed auxotroph
– Auxo = “increase”; troph = “nourishment”
• Prototroph does not require growth factors
– Proto = “first”
• Geneticists compare mutants to wild type
• Typical phenotype of strains isolated from nature
• For example, wild-type E. coli strain is prototroph
• Strains designated by three-letter abbreviations
– for example, Trp– cannot make tryptophan
– Streptomycin resistance designated StrR
8.2. Spontaneous Mutations
 Spontaneous mutations caused by normal processes
 Occur randomly at infrequent characteristic rates
• Mutation rate: probability of mutation each cell division
• Typically between 10–4 and 10–12 for a given gene
• Mutations passed to progeny
• Occasionally change back to original state: reversion
• Large populations contain mutants (for example, cells in
colony)
• Environment selects cells that grow under its conditions
•
•
•
•
For example, antibiotics select for resistant bacteria if present
Environment does not cause mutations
Single mutation rare; two even rarer
Physicians may use two antibiotics to reduce resistance
– Chance is product of mutation rate for each
8.2. Spontaneous Mutations
 Base substitution
most common
• Incorrect nucleotide
incorporated during
DNA synthesis
• Point mutation is
change
of a single base pair
8.2. Spontaneous Mutations
 Base substitution leads to three possible
outcomes
• Silent mutation: wild-type amino acid
• Missense mutation: different amino acid
• Resulting protein may only partially function
– Termed leaky
• Nonsense mutation:
Specifies stop codon
– Yields shorter protein
8.2. Spontaneous Mutations
 Base substitutions
• A mutation that inactivates gene is termed a null or
knockout mutation
• “Silent mutation” sometimes used to indicate mutation
that does not alter protein function
• Base substitutions more common in aerobic
environments
• Reactive oxygen species (ROS) produced from O2
• Can oxidize nucleobase guanine
– DNA polymerase often mispairs with adenine
8.2. Spontaneous Mutations
 Deletion or addition of nucleotides
• Impact depends on number of nucleotides
• Three pairs changes one codon
• One amino acid more or less
• Impact depends on location
within protein
• One or two pairs yields
frameshift mutation
• Different set of codons translated
• Often results in premature
stop codon
• Shortened, nonfunctional protein
• Knockout mutation
8.2. Spontaneous Mutations
 Transposons (jumping genes)
• Can move from one location to another
• Process is transposition
• Gene insertionally inactivated
• Function destroyed
• Most transposons have transcriptional terminators
• Blocks expression of downstream genes in operon
8.2. Spontaneous Mutations
 Transposons (jumping genes) (continued…)
• Classic studies carried out by Barbara McClintock
• Observed color variation in corn kernels resulting from
transposons moving into and out of genes controlling
pigment synthesis
8.3. Induced Mutations
 Induced mutations result from outside influence
• Agent that induces change is mutagen
• Geneticists may use mutagens to increase mutation rate
• Two general types: chemical, radiation
8.3. Induced Mutations
 Chemical mutagens may cause base substitutions
or frameshift mutations
 Some chemicals modify nucleobases
• Change base-pairing properties
• Increase chance of incorrect nucleotide incorporation
• Nitrous acid (HNO2) converts cytosine to uracil
– Base-pairs with adenine instead of guanine
8.3. Induced Mutations
 Some chemicals modify nucleobases (cont…)
• Alkylating agents add alkyl groups onto nucleobases
• Nitrosoguanidine adds methyl group to guanine
– Base-pairs with thymine
8.3. Induced Mutations
 Base analogs resemble nucleobases
• Have different hydrogen-bonding properties
• Can be mistakenly incorporated by DNA
polymerase
• 5-bromouracil resembles
thymine, often base-pairs
with guanine
2-amino purine resembles
adenine, often pairs with
cytosine
8.3. Induced Mutations
 Intercalating agents cause frameshift mutations
• Flat molecules that intercalate (insert) between adjacent
base pairs in DNA strand
• Pushes nucleotides apart, produces space
• Causes errors during replication
• If in template strand, a base pair added to synthesized
strand
• If in strand being synthesized, a base pair deleted
• Often results in premature stop codon
• Ethidium bromide is common intercalating agent
• Likely carcinogen
• Chloroquine, used to treat malaria, is another example
8.3. Induced Mutations
 Transposition
• Transposons can be used to generate mutations
• Transposon inserts into cell’s genome
• Generally inactivates gene into which it inserts
8.3. Induced Mutations
 Radiation: two types
• Ultraviolet irradiation forms thymine dimers
• Covalent bonds between adjacent thymines
– Cannot fit into double helix; distorts molecule
– Replication and transcription stall at distortion
– Cell will die if damage not repaired
– Mutations result from cell’s SOS repair mechanism
• X rays cause single- and
double-strand breaks in DNA
– Double-strand breaks
often produce lethal
deletions
• X rays can alter nucleobases
8.4. Repair of Damaged DNA
 Enormous amount of spontaneous and mutageninduced damage to DNA
• If not repaired, can lead to cell death; cancer in animals
• For example, in humans, two genes associated with breast
cancer code for DNA repair enzymes; mutations in either
result in 80% probability of breast cancer
• Mutations are rare; alterations in DNA generally repaired
before being passed to progeny
• Several different DNA repair mechanisms
8.4. Repair of Damaged DNA
8.4. Repair of Errors in Nucleotide Incorporation
 During replication, DNA polymerase sometimes
incorporates wrong nucleotide
• Mispairing slightly distorts DNA helix
• Recognized by enzymes
• Mutation prevented by repairing before DNA replication
• Two mechanisms: proofreading, mismatch repair
• Proofreading by DNA polymerase
•
•
•
•
Verifies accuracy
Can back up, excise nucleotide
Incorporate correct nucleotide
Very efficient but not flawless
8.4. Repair of Errors in Nucleotide Incorporation
• Mismatch Repair
• Fixes errors missed by DNA
polymerase
• Enzyme cuts sugarphosphate backbone
• Another enzyme degrades
short region of DNA strand
• Methylation of DNA indicates
template strand
– Methylation takes time,
so newly synthesized
strand is unmethylated
• DNA polymerase, DNA ligase
make repairs
8.4. Repair of Modified Nucleobases in DNA
 Modified nucleobases lead to
base substitutions
• Glycosylase removes
oxidized nucleobase
• Another enzyme cuts
DNA at this site
• DNA polymerase
removes short section;
synthesizes replacement
• DNA ligase seals gap
8.4. Repair of Thymine Dimers
 Several methods to repair damage from UV light
• Photoreactivation: light repair
• Enzyme uses energy from light
• Breaks covalent bonds of
thymine dimer
• Only found in bacteria
• Excision repair: dark repair
• Enzyme removes damage
• DNA polymerase, DNA ligase
repair
Repair of Thymine Dimers
 Several methods to repair damage from UV light
(continued…)
• SOS repair: last-ditch repair mechanism
•
•
•
•
Induced following extensive DNA damage
Photoreactivation, excision repair unable to correct
DNA and RNA polymerases stall at unrepaired sites
Several dozen genes in SOS system activated
– Includes a DNA polymerase that synthesizes even in
extensively damaged regions
– Has no proofreading ability, so errors made
– Result is SOS mutagenesis
8.5. Mutant Selection
 Mutants rare, difficult to isolate
• Two main approaches
• Direct selection: cells inoculated onto medium that
supports growth of mutant but not parent
• for example, antibiotic-resistant mutants exposed to
antibiotic
• Indirect selection: isolates
auxotroph from prototrophic
parent strain
• More difficult since parents will
grow on any media on which
auxotroph can grow
• Replica plating
8.5. Mutant Selection
8.5. Mutant Selection
 Penicillin enrichment of mutants sometimes
used
• Increases proportion of auxotrophs in broth culture
• Penicillin kills growing cells
• Prototrophs
• Auxotrophs survive
• Penicillinase then added
• Cells plated on rich medium
8.5. Mutant Selection
 Screening for Possible Carcinogens
• Carcinogens cause many cancers; most are mutagens
• Animal tests expensive, time-consuming
• Mutagens increase low frequency of spontaneous
reversions
• Ames test measures effect of chemical on reversion
rate of histidine-requiring Salmonella auxotroph
• Uses direct selection
• If mutagenic, reversion rate increases relative to control
• Rat liver extract added since non-carcinogenic chemicals
often converted to carcinogens by animal enzymes
• Additional tests on mutagenic chemicals to determine if
carcinogenic
8.5. Mutant Selection
Horizontal Gene Transfer as a Mechanism
of Genetic Change
 Microorganisms commonly acquire
genes from other cells: horizontal gene
transfer
• Can demonstrate recombinants
with auxotrophs
• Combine two strains
– for example, His–, Trp– with Leu–, Thr–
• Spontaneous mutants unlikely
• Colonies that can grow on
glucose-salts medium most likely
acquired genes from other strain
Horizontal Gene Transfer as a Mechanism
of Genetic Change
 Genes naturally transferred by three mechanisms
• Transformation: naked DNA uptake by bacteria
• Transduction: bacterial DNA transfer by viruses
• Conjugation: DNA transfer during cell-to-cell contact
Horizontal Gene Transfer as a Mechanism
of Genetic Change
 DNA replicated only if replicon
• Has origin of
replication
• Plasmids,
chromosomes
• DNA fragments
added to
chromosome via
homologous
recombination
• Only if sequence
similar to region
of recipient’s
genome
8.6. DNA-Mediated Transformation
• Naked DNA is not within
cell or virus
• Cells release when lysed
• Addition of DNase
prevents transformation
• Demonstration of
transformation in
pneumococci
• Only encapsulated cells
pathogenic
8.6. DNA-Mediated Transformation
 Transformation
• Recipient cell must be competent
• Most take up regardless of origin
• Some accept only from closely
related bacteria (DNA sequence)
• Process tightly regulated
• Bacillus subtilis has twocomponent regulatory system
• Recognizes low nitrogen or
carbon
• High concentration of bacteria
(quorum sensing)
• Only a fraction of population
becomes competent
8.7. Transduction
 Transduction: transfer of genes by
bacteriophages
• Specialized transduction: specific genes (Chapter 13)
• Generalized transduction: any genes of donor cell
• Rare error
during phage
assembly
• Transfer of
DNA to new
bacterial host
8.8. Conjugation
 Conjugation: DNA transfer between bacterial cells
• Requires contact between donor, recipient cells
• Conjugative plasmids direct their own transfer
• Replicons
• F plasmid (fertility)
of E. coli most
studied
• Other plasmids encode
resistance to some antibiotics
• Spread resistance easily
8.8. Conjugation
 Conjugation (continued…)
• F plasmid of E. coli
• F+ cells have, F– do not
• Encodes proteins including
F pilus
•
•
•
•
•
Sex pilus
Brings cells into contact
Enzyme cuts plasmid
Single strand transferred
Complementary strands
synthesized
• Both cells are now F+
8.8. Conjugation
 Chromosomal DNA transfer
less common
• Involves Hfr cells (high
frequency of recombination)
• F plasmid is integrated into
chromosome via homologous
recombination
• Process is reversible
• F′ plasmid results when small
piece of chromosome is
removed with F plasmid DNA
• F′ is replicon
8.8. Conjugation
 Chromosomal DNA transfer less
common (continued…)
• Hfr cell produces F pilus
• Transfer begins with genes on one
side of origin of transfer of plasmid
(in chromosome)
• Part of chromosome transferred to
recipient cell
• Chromosome usually breaks before
complete transfer (full transfer would
take ~100 minutes)
• Recipient cell remains F– since
incomplete F plasmid transferred
8.9. The Mobile Gene Pool
 Genomics reveals surprising variation in gene
pool of even a single species
• Perhaps 75% of E. coli genes found in all strains
• Termed core genome of species
• Remaining make up mobile gene pool
• Plasmids, transposons, genomic islands, phage DNA
8.9. The Mobile Gene Pool
 Plasmids found in many bacteria and archaea
•
•
•
•
•
Some eukarya
Usually dsDNA with origin of replication
Generally nonessential; cells survive loss
Few to many genes
Low-copy-number
• One or a few per cell
• High-copy-number
• Many up to 500
• Most have narrow host range
• Single species
• Some broad host range
• Includes Gram– and Gram+
8.9. The Mobile Gene Pool
 Resistance plasmids (R plasmids)
• Resistance to antimicrobial medications, heavy
metals (mercury, arsenic)
• Compounds found in hospital environments
• Often two parts
• R genes
• RTF (resistance
transfer factor)
– Codes for conjugation
• Often broad host range
• Normal microbiota can
transfer to pathogens
Copyright © 2016 McGraw-Hill Education. Permission required for reproduction or display.
Use fig. 8.26 in 8th edition
8.9. The Mobile Gene Pool
 Transposons provide mechanism for moving DNA
• Can move into other replicons in same cell
• Simplest is insertion sequence (IS)
• Encodes only transposase enzyme, inverted repeats
• Composite transposons include one or more genes
• Integrate via non-homologous recombination
8.9. The Mobile Gene Pool
 Transposons yielded vancomycin resistant
Staphylococcus aureus strain
• Patient infected with S. aureus
• Susceptible to vancomycin
• Also had vancomycin resistant
strain of Enterococcus faecalis
• Transferred transposoncontaining plasmid to S. aureus
• Transposon jumped to
plasmid in S. aureus
8.9. The Mobile Gene Pool
 Bacteria can conjugate with plants
• Natural genetic engineering
• Agrobacterium tumefaciens causes crown gall
• Different properties, produces opine, plant hormones
• Piece of tumor-inducing (Ti) plasmid called T-DNA
(transferred DNA) is transferred to plant
• Incorporated into plant chromosome via nonhomologous recombination
8.9. The Mobile Gene Pool
 Genomic islands: large DNA segments in genome
• Originated in other species
• Nucleobase composition very different from genome
• G-C base pair ratio characteristic for each species
• May provide different characteristics
•
•
•
•
Utilization of energy sources
Acid tolerance
Development of symbiosis
Ability to cause disease
– Pathogenicity islands