Chapter 8 Flashcards
Barbara McClintock-what did she study
Studied color variation in corn kernels.
She noticed that the kernel colors were not inherited in a predictable manner.—> colors varied from one ear of corn to another.
What did McClintock conclude based on extensive data
McClintock concluded that segments of DNA, now called transposons were moving into and out of genes involved with kernel color.
What did the moving DNA segments do?
These moving DNA segments destroyed the function of the genes, thereby changing kernel color.
What did most scientists believe at the time McClintock published her results
At the time that McClintock published her results, most scientists believed that chromosomal DNA was very stable and changed only through recombination.
Where were transposons first discovered
In plants
Transposons are popularly called
Jumping genes
Natural selection
Selection by the environment of those cells best able to grow in that environment.
Bacteria have two general means by which they routinely adjust to new circumstances:
- Regulating gene expression
- Genetic change
What is a model used to study genetic change
To study genetic change, scientists often use E.coli as a model system.
Why is E.coli a good model for studying genetic change
- The cells grow rapidly in small volumes of simple, inexpensive media.
Strains
A pure culture isolate; genetic variant within a species
Genetic change in bacteria occurs by two mechanisms:
- Mutation
- Horizontal gene transfer
Mutation
Changes the existing nucleotide sequence of a cell’s DNA, which is then passed on to the progeny (offspring) through vertical gene transfer.
Vertical gene transfer
Transfer of genes from parent to offspring
Mutants
The modified organism and progeny are referred to as mutants
Horizontal gene transfer (HGT)
Is the transfer of DNA from one organism to another by a process other than reproduction
Like mutations, the changes are then passed on to the progeny by vertical transfer.
What happens to changes that occur in horizontal gene transfer
Like mutations, the changes are then passed on to the progeny by vertical transfer.
Genotype
The sequence of nucleotides in an organism’s DNA
A change in an organism’s DNA alters it’s
Genotype
In bacterial cells, a change in genotype can have a significant effect why?
In bacterial cells, such a change can have a significant effect because bacteria are haploid, meaning that they contain only a single set of genes.
Haploid
They contain only a single set of genes
Because a bacteria are haploid, a change in genotype…
A change in genotype often alters the organism’s observable characteristics or phenotype.
Phenotype
The observed characteristics of a cell.
Phenotype can be influenced by
Environmental conditions
Auxotroph
A mutant/microorganism that requires an organic growth factor
Prototroph
A microorganism that does not require any organic growth factors
Wild E.coli is what kind of troph
Wild type E.coli is a prototroph
What is “wild type” E.coli
Wild type refers to the typical phenotype of strains isolated from nature.
A strains characteristics are designated by:
Three letter abbreviations, with the first letter capitalized.
Spontaneous mutations
Mutation that occurs naturally during the course of normal cell processes
Are random genetic changes that result from normal cells processes and are passed on to a cell’s progeny.
Because spontaneous mutations occur routinely
Every large population contains mutants, so cells in a colony are not necessarily identical.
What happens to an organism that has spontaneously mutated to become resistant to an antimicrobial medication?
An organism that has spontaneously mutated to become resistant to an antimicrobial medication will become dominant in an environment where the medication is present because the sensitive cells are killed or inhibited, allowing the resistant cells to grow without competition.
The mutation rate of different genes usually varies between what
The mutation rate of different genes usually varies between 10^-4 and 10^-12 per cell division.
What is the chance that a gene will undergo a mutation when a cell replicated its DNA prior to cell division
Between one in 10,000 (10^-4) and one in a trillion (10^-12)
Reversion or back mutation
Mutation that corrects a defect caused by an earlier mutation
How is reversion similar to mutation
Like the original mutation, reversion occurs spontaneously at low frequencies.
What is the most common type of mutation
Base substitution
Base substitution
Occurs during DNA synthesis when a DNA polymerase incorporates an incorrect nucleotide
Point mutation
Mutation in which only a single base pair is involved
Base substitution in a protein encoding gene can lead to what three possible mutation outcomes:
- Synonymous mutation
- Missense mutation
- Nonsense mutation
Synonymous mutation
Incorporation of the incorrect nucleotide creates a codon that encodes the same amino acid as the original.
A mutation that does not change the amino acid encoded
Why can an incorrect nucleotide still encode the same amino acid as the original?
This can occur because of the redundancy of the genetic code; recall that most amino acids are coded for by more than one codon
What is another name for synonymous mutation
Silent mutation
Missense mutation
Incorporation of the incorrect nucleotide creates a codon that codes for a different amino acid.
A mutation that changes the amino acid encoded by DNA.
What impacts the effect of Missense mutation
The effect of this depends on the position and the nature of the change within the protein.
In many cases, what happens to cells with a Missense mutation
In many cases, cells with a Missense mutation grow slowly because the encoded protein does not function as well as normal.
Nonsense mutation
Incorporation of the incorrect nucleotide creates a stop codon.
A mutation that generates a stop codon, resulting in a shortened protein.
What is the result of a nonsense mutation
This results in a shorter truncated protein that is often non functional.
Null or knockout mutation
Any mutation that totally inactivates the gene is termed null or knock out mutation
What conditions are base substitutions most common for bacteria
Base substitutions are more common when bacteria are growing in aerobic environments, as opposed to anaerobic ones.
Why are base substitutions more common in aerobic conditions than anaerobic conditions
This is because reactive oxygen species (ROS) such as superoxide and hydrogen peroxide are produced from O2.
These chemicals can oxidize the nucleobase guanine, and DNA polymerases often mispair oxidized guanine with adenine rather than cytosine.
Examples of Reactive Oxygen Species (ROS)
- Superoxide
- Hydrogen peroxide
What do the ROS do
These chemicals can oxidize the nucleobase guanine, and DNA polymerases often mispair oxidized guanine with adenine rather than cytosine.
What can errors during DNA synthesis lead to
Errors during DNA synthesis can also lead to the deletion or addition of nucleotides.
The consequences of deletion or addition of nucleotides depends on what
The consequence depends on how many nucleotides are involved.
If three nucleotides are added (or deleted), what happens
This adds (or deletes) one codon
When the gene is expressed, one additional (or one less) amino acid will be in the resulting protein.
The seriousness of addition or subtraction of one amino acid in the resulting protein depends on what
The seriousness of the changes effect depends on its location in the encoded protein.
Adding or subtracting one or two nucleotide pairs causes what
Adding or subtracting one or two nucleotide pairs causes a frame shift mutation.
Frameshift mutation
Mutation resulting from the addition or deletion of a number of nucleotides not divisible by 3.
What does the frameshift mutation cause
This changes the reading frame of the corresponding mRNA molecule so that an entirely different set of codons is translated.
What is a frequent example of a frame shift mutation
Frequently, one of the resulting downstream codons will be a stop codon.
What is the end result or consequence of the common frame shift mutation example a stop codon
As a consequence, a frame shift mutation likely results in a shortened, nonfunctional protein a knockout or nonfunctional protein.
Transposons
Are pieces of DNA that can move from one location to another in a cell’s genome, a process called transposition.
Transposition
The process of pieces of DNA moving from one location to another in a cell’s genome.
Insertional inactivation
Disruption of the function of a gene due to a DNA segment inserted into the gene.
What is the outcome of insertional inactivation
The gene into which a transposon jumps is inactivated by the event.
What do most transposons contain
Most transposons contain transcriptional terminators, so the expression of downstream genes in the same operon will stop as well.
Induced mutations
Are genetic changes that occur due to an influence outside of a cell
Influences from outside the cell such as what can cause induced mutations
Exposure to chemical or radiation
Mutagen
An agent that induces genetic changes
Any agent that increases the frequency at which DNA is altered (mutated).
What do geneticists rely on to study cellular processes
Geneticists who depend on mutants to study cellular processes often use mutagens to increase the mutation rate in bacteria, making mutants easier to find.
What do geneticists use to increase the mutation rate in bacteria
Mutagens
Mutagenesis
Using mutagens to increase the mutation rate in bacteria.
Chemical mutagens can cause
- Base substitutions
- Frameshift mutations
What do different chemicals do to DNA
A number of different chemicals modify the nucleobases in DNA, changing their base pairing properties.
By doing so, the chemicals increase the chance that an incorrect nucleotide will be incorporated during DNA replication
What do chemicals called alkylating agents do
Chemicals called alkylating agents add alkyl groups ( a type of functional group) onto nucleobases;
What happens when the alkylating agent nitrosoguanine adds a methyl group to guanine
The modified nucleobase sometimes base pairs with thymine.
Base analog
A compound that structurally resembles a nucleobase closely enough to be incorporated into a nucleotide in place of the natural nucleobase.
How do base analogs differ from natural nucleobases
They have different hydrogen bonding properties.
What happens when the analogs are mistakenly used
The analogs can be mistakenly used in place of the nucleobases when the cells make nucleotides, and DNA polymerases then incorporate these into DNA.
What happens to the complementary strand when the analog is used
When the complementary strand is synthesized, the wrong nucleotide may be incorporated opposite the base analog.
Intercalculating agents
Agents that insert between base pairs in a DNA double helix
What do intercalculating agents cause
Intercalculating agents increase the frequency of Frameshift mutations
What do Intercalculating agents increase the chance of
Intercalculating agents insert between adjacent bases of DNA, pushes nucleotides apart, producing a space between bases and thereby increasing the chance that insertions or deletions will be made during DNA replication.
What does Intercalculating agents result in
This often results in the premature generation of a stop codon, giving rise to a shortened protein.
Example of Intercalculating agents
Chemicals used to stain DNA in the lab
Why can’t transposons replicate on its own
Because it lacks an origin of replication
Two kinds of radiation commonly used as mutagens
- Ultraviolet light
- X rays
How does UV light effect DNA strand
UV light exposure causes covalent bonds to form between adjacent thymine nucleobases on a DNA strand producing thymine dimers.
Thymine dimers
Two adjacent thymine molecules on the same strand of DNA joined together through covalent bonds
What do dimers do
Dimers distort the DNA molecule because they cannot fit properly into the double helix.
Replication and transcription stop at the distortion, and as a result, the cells will die if the damage is not repaired.
How do dimers affect replication and transcription of DNA
Replication and transcription stop at the distortion and as a result, the cells will die of the damage is not repaired.
X rays affect of DNA
X rays cause single and double strand breaks in DNA.
What do double stranded breaks often cause
Double stranded breaks often result in deletions that are lethal to the cell.
How common are mutations and why
Mutations are rare because cells have multiple mechanisms to repair damaged DNA before errors are passed on to progeny.
What types of DNA damage cannot be repaired
Insertional inactivation caused by transposons cannot be repaired.
What errors do DNA polymerase do and what does this result in
DNA polymerase sometimes incorporate the wrong nucleotide as they synthesize DNA.
The resulting mispair info of nucleobases results in a slight distortion in the DNA helix, which can be recognized by enzymes within the cell that then repair the mistake.
What does the errors caused by DNA polymerase result in?
The resulting mi
By quickly repairing the error before DNA is replicated, what can happen
By quickly repairing the error before the DNA is replicated, the cell prevents the mutation.
What are the two mechanisms for repairing error in DNA
- Proofreading by DNA polymerase
- Mismatch repair
Proofreading
The detection and removal by DNA polymerase of an incorrect nucleotide incorporated as DNA is synthesized.
How do DNA polymerase proofread DNA
The enzymes can back up and excise (remove) a nucleotide not correctly hydrogen bonded to the opposing nucleobase in the template strand.
The DNA polymerase then inserts the correct nucleotide.
Mismatch repair
Fixes errors missed by the proofreading of DNA polymerases.
Repair mechanism in which enzyme cuts the DNA near a mismatched nucleobase, resulting in the removal and replacement of a short stretch of nucleotides.
How does mismatch repair work
A specific protein binds to the site of the mismatched nucleobase, directing an enzyme to cut the sugar phosphate backbone of the new DNA strand.
Another enzyme then degrades a short region of that DNA strand, thereby removing the misincorporated nucleotide.
During mismatch repair, how do the enzymes know which is the template strand and which is the new strand
Soon after a DNA strand is synthesized, an enzyme adds methyl groups to certain nucleobases. This takes time, so the new strand is still unmethylated immediately after it is synthesized. Because the template strand is methylated, whereas the new strand is not, the repair enzyme can distinguish between the two.
What happens after the mismatch repair is complete
After the nucleotides are removed from the new strand, the combined actions of a DNA polymerase and DNA ligase then fill in that section and seal the gap.
Base excision repair
A mechanism cells use to remove damaged nucleobases in DNA and then repair the region of damage.
What enzyme is involved in base excision repair
DNA glycosylase
How does base excision repair work
DNA glycosylase removes damaged nucleobase from the sugar phosphate backbone.
Another enzyme then recognizes that a nucleobase is missing and cuts the DNA at this site.
A DNA polymerase degrades a short section of this strand to remove the damage.
The same enzyme synthesizes another star and with the proper nucleotides, and DNA ligase seals the gap in the single stranded DNA.
Mechanisms used by bacteria to repair damage by UV light
- Photoreactivation
- Nucleotide excision repair
Photoreactivation another name for it
Light repair
How does Photoreactivation work
Relies on an enzyme that uses the energy of visible light to break the covalent bonds of the thymine dimer. By breaking the bond, the DNA is restored to its original state.
Another name for nucleotide excision repair
Dark repair
How does nucleotide excision repair work
Does not require energy from visible light.
A specific enzyme recognizes the major distortions in DNA that result from thymine dimer formation and then removes the DNA strand with the damaged region.
A DNA polymerase and DNA ligase then fill and seal the gap left by the removal of the segment.
SOS repair
Complex, Inducible and error prone process used to repair highly damaged DNA
When are the enzymes that carry out SOS repair induced
The enzymes that carry out SOS repair are induced when DNA is so heavily damaged that other repair systems may not be able to correct all the damage.
How does SOS repair work
DNA and RNA polymerases stall at sites of unrepaired damage, so the cells cannot replicate or transcribe their DNA.
Damaged DNA activates the expression of the several dozen genes that encode the SOS system.
One component of this system is a DNA polymerase that synthesizes DNA even in extensively damaged regions.
What is the result of the SOS polymerase having no proofreading ability
Errors are made as a result, a process called SOS mutagenesis.
How do standard DNA polymerases differ from SOS DNA polymerase
The SOS DNA polymerase has no proofreading ability.
Direct selection
Technique of selecting mutants by plating organisms on a medium on which the desired mutants but not the parent will grow.
Example of direct selection
Antibiotic resistant mutants can be easily selected directly by inoculating cells onto a medium containing the antibiotic. Only the resistant cells will form colonies.
Indirect selection
A technique for isolating mutants and identifying organisms unable to grow on a medium on which the parents do grow; often involves replica plating.
What is indirect selection used to isolate
Is used to isolate an autotrophic mutant from a prototrophic parent strain.
Why is indirect selection more difficult than direct selection
Because no medium allows growth of autotrophs but not prototrophs
What does indirect selection often require
Indirect selection generally involved a method called replica plating.
Replica plating
A method for indirect selection that involves the simultaneous transfer of organisms in separated colonies from one medium to another.
Who developed replica plating
Joshua and Esther Lederberg
Steps for replica plating
Reading on pg 84.
Penicillin enrichment
Pg 84
Horizontal gene transfer
How microorganisms commonly acquire genes from other cells.
Allows organisms to change and adapt.
Recombinant
A cell that carries a DNA molecule derived from two different DNA molecules
Recombinants are a result of HGT.
Genes can be transferred from donor to recipient by three different mechanisms:
- Bacterial transformation
- Transduction
- Conjugation
Bacterial transformation
Naked DNA is taken up from the environment by a bacterial cell.
Transduction
DNA is transferred from one bacterial cell to another by bacteriophage (a virus that infects bacteria)
Mechanism of HGT in which bacterial DNA is transferred inside a phage coat.
Bacteriophage
A virus that infects bacteria
Also called a phage
Conjugation
DNA is transferred during cell to cell contact
What must occur following gene transfer
A recipient cell must replicate the DNA to pass it on to daughter cells.
Replicon
Piece of DNA that contains an origin of replication and therefore can potentially be replicated by a cell.
Examples of replicons
- Chromosomes
- Plasmids
Homologous recombination
Process by which a cell replaces a stretch of DNA with a segment that has a similar nucleotide sequence.
How does homologous recombination work
The donor DNA becomes positioned next to the complementary region of the recipient cells DNA.
The donor DNA then replaces a homologous segment of recipient DNA and the DNA it replaced is degraded.
Frederick Griffith experiment with mice
Pg 85
Bacterial transformation is also referred to as
DNA mediated transformation or simple transformation
What is naked DNA
Naked DNA is DNA that is free in the cell’s surroundings; it is not contained within a cell or a virus.
Where do naked DNA usually come from
Naked DNA often originates from cells that have burst.
Another source of naked DNA is certain bacterial species that secrete small pieces of DNA, presumably as a means of promoting transformation.
DNAse
Enzyme that degrades DNAs
What type of DNA can DNAse only degrade
DNase will destroy only DNA that is free in the medium; it cannot access/degrade DNA within a cell or a phage.
Competent
In horizontal gene transfer, physiological conditions in which bacterial cell is capable of taking up DNA.
Simply, how does bacterial transformation work
In bacterial transformation, DNA is released from donor cells and taken up by competent recipient cells.
Competent cells bind DNA and take up a single strand; that strand then integrates into genome by homologous recombination.
How do bacterial viruses (phases) transfer bacterial genes from a donor to a recipient
Transduction
What are the two types of transduction
- Generalized
- Specialized
What do phages consist of
- DNA or RNA
- Surrounded by a protein coat.
How does a phage infect a bacterium
A phage infects a bacterium by attaching to the cell and then injecting its nucleic acid into that cell.
Enzymes encoded by the phage genome then cut the bacterial DNA into small pieces.
Next, the bacterial cells enzymes replicate the phage nucleic acid and synthesize proteins that make up the phage coat and the various components assemble to produce complete phage particles
These new phage particles are released from the bacterial cell, usually as a result of host cell lysis.
Generalized transduction
Results from a rare error that sometimes occurs during the construction of phage particles.
A fragment of bacterial DNA mistakenly enters the phage protein coat. This error creates what is called transducing particle.
What does generalized transduction often produce
A transducing particle
Transducing particle
Bacteriophage progeny that contains part of a bacterial genome instead of phage DNA due to an error during packaging.
Carries no phage DNA and therefore insert not a phage.
What does the transducing particle do once made
Like phage particles, a transducing particle will attach to another bacterial cell and inject the DNA it contains. The transducing particle injects only bacterial DNA because that is all it contains.
The bacterial DNA may then integrate into the recipients chromosome by homologous recombination.
Conjugative plasmids
Plasmid that carries the genes for a sex pilus and can transfer copies of itself to other bacteria during conjugation.
What is the most thoroughly studied conjugative plasmid
F plasmid
F plasmid
Plasmid found in donor cells of E.coli that codes for the F or sex pilus and makes the cell F+
F pilus or sex pilus
A protein appendage used for DNA transfer in the process of conjugation
What are the four steps involved in plasmid transfer
- Making contact
- Initiating transfer
- Transferring DNA
- Transfer complete
What occurs during the 1st step in plasmid transfer
- Making contact
The F pilus of the donor cell binds to a specific receptor on the outer membrane of the recipient.
What occurs in the second step of plasmid transfer
Initiating transfer
After contact, the F pilus retracts, pulling the two cells together.
Meanwhile a plasmid encoded enzyme cuts one strand of the F plasmid at a specific nucleotide sequence, the origin of transfer.
What is the third step of plasmid transfer
Transferring DNA
A single strand of F plasmid enter the F- cell.
Once inside the recipient cell, that strand serves as a template for synthesis of the complementary strand, generating the F plasmid.
What is the fourth step of plasmid transfer
Transfer complete
Both the donor and the recipient cells are now F+ so they can act as donors of the F plasmid.
Hfr cells
Cells that have the F plasmid integrated into their chromosome, allowing them to begin transferring the chromosome by conjugation; stands for high frequency of recombination cells
What does chromosomal DNA transfer involve
Chromosomal DNA involves Hfr cells.
Crown gall
Pg 88
