Download - [Micro] bacterial genetics (6 jan)
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Bacteria use three main mechanisms to adapt to
changing environments
– Mutation
– Gene transfer
– Regulation of gene expression
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Mutation
Spontaneous mutations occur infrequently
and randomly in the natural environment
Rate of spontaneous mutation
Probability that a mutation will be observed in a given
gene each time the cell divides
Rate is generally between 1 in 10,000 and 1 in a trillion
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Mutations are stable heritable changes inthe base sequence of DNA
Mutations can be caused by:
Base substitutions
Removal or addition of nucleotides
Transposable elements
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Base substitutionsMost common type of mutation
Results from mistakes during DNA
replication
Point mutations: when one base pair is
changed
Missense mutation
Nonsense mutation: Mutation that
changes an amino acid codon to a stop
codon
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Removal and addition of nucleotidesShifts the translational reading frame
Called frameshift mutation because it affects all amino acids downstream from addition or deletion (frequently result in premature stops)
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Transposable elements
Special segments (transposons) of DNA that move
spontaneously from one gene to a different gene
Transposons may disrupt the integrity of the gene and
render its protein product nonfunctional
Read about Barbara McClintock (A Glimpse of History) on pg. 191
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Mutations are essential for understanding genetics
Mutations can be intentionally produced (induced
mutations) to demonstrate function of particular gene or
set of genes
Mutations can be induced via:
Chemical mutagens
Transposition
Radiation
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Chemical mutagens
Nitrous acidChanges cytosine to uracil
Alkylating agents
Alter hydrogen bonding of bases
Nitrosoguanine is common alkylating agentUsed as antineoplastic drugs
Base analogs
Chemicals that are structurally similar to the nitrogenous bases buthave slightly altered base pairing properties
Base analogs include:2-aminopurine which incorporates in the place of adenine butbinds with cytosine
5-bromouracil which incorporates in the place of thymine but binds with guanine
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Intercalating agents
Molecules that insert themselves between adjacent
bases and create space between bases
Ethidium bromide is common intercalating agent
Potential carcinogen
Used extensively in biochemical/molecular biological
research
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Transposition
Common procedure used to induce mutation in thelaboratory
A transposon inserts into a gene (insertion mutation)
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Radiation
Ultraviolet light
Causes covalent bonding between adjacent thymine bases forming thymine dimers which distort DNA
X rays
Cause breaks and alterations in DNA. Breaks that occur on both strands are often lethal
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Mutations and Their Consequences
Mutations provide the organism with a way to respond to environmental stresses
Environment selects for cells suited to survive
Environment does not cause mutation
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WESTERN BLOT
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WESTERN BLOT
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WESTERN BLOT
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WESTERN BLOT
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NORTHERN BLOT
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NORTHERN BLOT
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NORTHERN BLOT
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NORTHERN BLOT
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SOUTHERN BLOT
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Southern blot
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Mechanisms of Gene Transfer
Genes are naturally transferred between bacteria
DNA-mediated transformation
Transduction
Conjugation
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Gene exchange in bacteria
1. Donor DNA is transferred and accepted by the recipient cell
Three mechanisms
DNA-mediated transformation
Transduction
Conjugation
2. Donor DNA is integrated into the recipient cell’s chromosome
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No integration
Integration
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DNA-mediated transformation (transformation)
The transfer of naked DNA from one bacterium toanother
Discovered by Fredrick Griffith in 1928 while working with Streptococcus pneumoniae
Griffith realized S. pneumoniae existed in two formsEncapsulated, virulent form (smooth in appearance)Nonencapsulated, avirulent form (Rough in appearance)
Griffith hypothesized that injections with the smoothstrain could protect mice from pneumonia
Griffith injected mice with the two different strains
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Griffith’s Results
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Natural transformation occurs when bacterial
cells are “competent”
Competence is a condition in which bacterial cells
are capable of taking up and integrating large
fragments of DNA into their chromosome
Competence usually occurs naturally during the late log,
early stationary phase
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Dying cells rupture during the stationary and death phases. The chromosome breaks into small pieces and explodes through the ruptured
cell wall
Recipient cells absorb pieces of “naked” DNA
The naked DNA is integrated into the recipient
cell’s chromosome
Naked DNA integrates at a homologous site on the recipient’s
chromosome
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Entry of the DNAOnly single strands enter
Integration of the donor DNADonor DNA is integrated at ahomologous site
Enzymes cleave recipient DNA
Donor DNA replaces recipient DNAvia recombination
Cell multiplicationTransformed cells multiply under selectiveconditions in which non transformed cellswill not grow
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Transduction
Bacterial DNA that is transferred from donor to
recipient via a bacterial virus (bacteriophage)
Two types of transductionGeneralized
Any gene from the donor can be transferred
SpecializedOnly specific genes can be transferred
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Transduction
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Conjugation
Conjugation is mediated by a plasmid R plasmids
F plasmids
Conjugation requires direct contact between cells
Cells must be of opposite mating types
Donor cells carry a plasmid that codes for fertility factor or “Ffactor”
This cell is designated F+
Recipient cell does not carry a plasmidThis cell is designated F-
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Conjugation
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High frequency of recombination – Hfr strains