Post on 02-Jun-2018
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Ch. 7. Microbial Genetics
Learning Objectives: You should be able to explain anddescribe the following:DNA structure and compositionPlasmidsDNA replicationHow DNA codes for proteins (transcription and translation)RNA structure and compositionTypes of mutations (point and frameshift)Mutagens
Methods for selecting mutantsThe Ames TestGenetic recombination and gene transfer (transformation,
conjugation, transduction, and transposition)
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DNA structure and composition
The basic unit of DNA is the nucleotide thatcontains a phosphate group, deoxyribose sugar,
and a nitrogenous base
In the helix the strands are orientedantiparallel, base-pairing occurs in the center(hydrophobic region), with a hydrophilicphosphate-sugar backbone on the outside
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The bidirectionality of DNA replication
Origin
Replicationproceeds in bothdirections
Replication forks
Terminationof replication
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Transcription
Translationby ribosomes
5
5
5
3
3
3
DNA(genotype)
mRNA
Polypeptide(made ofamino acids)
Phenotype
NH 2 Methionine Arginine Tyrosine Leucine
reading framefor theribosomes
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Genetic Change in Bacteria
A genetic change alters an organism
s genotype
Bacteria are haploid
- have only 1 copy of their DNA, therefore achange in DNA can easily alter thephenotype of the bacteria
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Mutations of Genes
Mutation change in the nucleotide base sequence
Almost always harmful to the bacterial cell
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Types of Mutations
Point mutations one base pair is affected
E.g.: insertions, deletions, and substitutions
Frameshift mutations nucleotide triplets afterthe mutation are displaced
E.g.: insertions and deletions
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Point mutations
Changes in one base pair can lead to one of 3 possibilities:1. silent mutation no change in amino acid sequence
2. missense mutation change in amino acid sequence
3. nonsense mutation premature termination of the
protein synthesis
usually defective protein
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Is the mutated protein s function likely to be altered?
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Is the mutated protein
s function likely to be altered?
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Is the mutated protein
s function likely to be altered?
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Frameshift mutations
Ribosomes
read
the mRNA in codons(groups of 3 nucleotides); this is called areading frame
When frameshift mutations occur, these lead toa shift in the reading frame used by theribosomes, often leading to changes in theamino acid sequence of the protein
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Is the mutated protein s function likely to be altered?
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Ultraviolet light
Thymine dimer
Mutagens
Ionizing radiation induces breaksin chromosomes
Nonionizingradiation induces
pyrimidine
dimers(e.g. thymine
dimers)
What are the effects of thymine dimers on DNA?
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Mutagens
One example of a chemical mutagen
Nucleotide analog (e.g. base analog)
structurally similar to a normal nucleotide, may be
incorporated into DNA in place of a normal
nucleotide; it disrupts DNA and RNA replication
and causes point mutations
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An example of a base analog is 5-bromouracil which incorporates in place of thymine and binds withguanine
This will change the complementary strand beingsynthesized during DNA replication
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i i l i
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Positive selectionof mutants
In this example weare looking forcells that afterexposure to amutagen, havemutated and
become resistantto penicillin, and
can grow inmedium with
penicillin
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Stamp replica plateswith velvet.
Bacteria
Identify auxotrophas colony growing oncomplete medium butnot on lacking medium.
Inoculate auxotrophcolony into completemedium.
Complete mediumcontaining tryptophan
Medium lackingtryptophan
Incubation
All colonies grow. Tryptophan auxotrophcannot grow.
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The Ames Test Liver extract is used to mimic what happens in the human
liver (our liver helps us detoxify compounds, sometimesleading to the production of carcinogenic compounds)
A particular type of Salmonella mutant is used. This mutant
is his-
which means it cannot grow without the addition ofthe amino acid histidine in the medium
The goal of this assay is to test whether a chosen chemical iscapable of causing a mutation (i.e. capable of being amutagen) and causes the Salmonella mutant to revert its his
-
mutation and become his+
(his + means that the Salmonella is able to grow in medium without the addition of the amino
acid histidine).
Th
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TheAmesTest
Liverextract
Liverextract
Suspectedmutagen
Experimentaltube
Controltube
Culture ofhis Salmo nella
Mediumlacking
histidine
Incubation
Colony of revertant ( h is + ) Salmonel la No growth
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Genetic Recombination and Transfer
Homologous recombination: exchange of nucleotide sequences (homologous sequences)
Recombinants cells with DNA molecules that contain newnucleotide sequences
Vertical gene transfer organisms replicate theirgenomes and provide copies to descendants
Genetic
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Geneticrecombination Homologous
sequences
Enzyme nicks one strand ofDNA at homologous sequence.
3
5
3
5
DNA A
DNA B
B
A
Recombination enzyme inserts the cut strand intosecond molecule, which is nicked in the process.
Ligase anneals nicked endsin new combinations.
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Molecules resolveinto recombinants.
Recombinant A
Recombinant B
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Horizontal Gene Transfer Among Prokaryotes
Three types of horizontal gene transfer:
Transformation Transduction Bacterial conjugation
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Horizontal Gene Transfer Among Prokaryotes
Transformation
Cells that take up DNA are competent
Transformation in
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Transformation inStrep tococ cus pneumo niae
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Horizontal Gene Transfer Among Prokaryotes
Transduction
A virus (phage) carries DNA segment from donor
cell to recipient cell
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Phage injectsits DNA.
Phage enzymesdegrade host DNA.
Phage DNA
Bacterialchromosome
Cell synthesizes new
phages that incorporatephage DNA and, mistakenly,some host DNA.
Transducing phage
Phage
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Transducing phage
Transducing phageinjects donor DNA.
Recipient host cell
Donor DNA is incorporatedinto recipient
s chromosomeby recombination.
Transduced cell
InsertedDNA
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Conjugation is another way to transfer DNA betweenneighboring cells (horizontal transfer)
Bacterial conjugation
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j g
F plasmid Origin oftransfer
Conjugation pilus
Chromosome
F+ cell F cell
Donor cell attachesto a recipient cellwith its pilus.
Pilus may drawcells together.
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Donor chromosome Pilus
Pilus
F+ cell
Hfr cell
F recipient
F plasmid integrates
into chromosome byrecombination.
Cells join via aconjugation pilus.
Formation of an Hfr (High frequency of recombination cell) and whathappens when it encounters a F- cell:
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F+ cell (Hfr)
F plasmidPart of F plasmid
Donor DNA Portion of F plasmid partiallymoves into recipient cell.
Conjugation ends with piecesof F plasmid and donor DNAin recipient cell.
Donor DNA and recipientDNA recombine, making a
recombinant F
cell.
Incomplete F plasmid;cell remains F
Recombinant cell (still F )
Does this process require cell cell contact? How does this affectthe size of the DNA that is transferred from the donor to therecipient cell?
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Transposons and Transposition
Transposons are segments of DNA that move from one
location to another in the same or different molecule Result is a frameshift insertion