nonselectable mutations

those that usually have neither an advantage nor a disadvantage over the parent

- maybe changed the metabolic pathway slightly but the cell growth just fine

detecting such mutations requires examining a large # of colonies and looking fr differences (screening)

screening basically means ___

hard process where you have to examine thousands of colonies individually to find the one with the subtle change you're looking for

its tedious compared to the selection

one classic screening methods involve ____

finding nutritional auxotroph using negative selections and replica plating

replica plating is ___

one of the methods available to facilitate screening

it is useful for identifying cells with a nutritional requirement for growth (auxotroph)

auxotroph is ___

a mutant that has lost its ability to make some essential nutrients so it now requires that nutrient in its growth medium

so it needs help.

screening for nutritional auxotrophs process:

so the process starts w a master plate:

1. you grow your mix of potentially mutated cells on complete medium one that has everything a cell can possibly need
2. so all colonies grow wildtype and potential auxotrophs alike (everythings happy there)
3. then comes the replicating plating itself, you use something like sterile velvet or toothpicks to pick up a pattern of colonies from the master plate and transfer that exact pattern onto two new plates simultaneously
4. MAINTAINING THE LAYOUT IS KEY
5. one new plate is another complete medium plate (this is our control) all the colonies you transferred should grow here, proving the transfer worked
6. the other plate has a selective medium, specifically lacking the nutrient that the hypothetical auxotroph cant make, so the auxotroph wont be able to grow there
7. so you compare the control plate to the selective plate
8. the missing colonies tells exactly which one is the auxotroph, so you can go back to the master plate and pick that specific colony
9. its process of elimination

induced mutations

those made environmentally or deliberately

can result from exposure to natural radiation or oxygen radicals

spontaneous mutations

those that occur without external intervention (usually mistake during DNA replication, or DNA damage, too many radicals, etc)

point mutations

mutations that change only one base pair (one nucleotide in genome that changed)

can lead to single amino acid change in a protein, an incomplete protein, or no change at all

cells dont have any ____

resistance

reason for mutation?

antibiotic is a selection pressure, mutation is random

what factor promotes mutation?

mutagen, chemicals, and viruses that damage DNA

silent mutation

does not affect amino acid sequence (nucleotide sequence change but no amino acid changes)

missense mutation

amino acid changed; polypeptide altered (nucleotide sequence change and amino acid changes)

nonsense mutation

codon becomes stop codon; polypeptide is incomplete (coding codon->stop codon)

types of mutation (photo)

deletions and insertions cause more ____

dramatic changes in DNA (frameshift mutation)

frameshift mutations

deletions or insertions that result in a shift in the reading frame

often result in complete loss of gene function

point mustions are typically ___

reversible

reversion

alteration in DNA that reverses the effects of a prior mutation (second mutation corrected the first mutation)

revertant

strain in which original phenotype is restored

(there are two types)

a type of revertant: same site revertant

mutation is at the same site as original mutation

a type of revertant: second-site revertant

(the same as suppressor)

mutation is at a different site in the DNA

suppressor

mutation that compensates for the effect of the original mutation

reversion and suppressor (photo)

for most microorganisms, errors in DNA replication occur at a frequency of ____

10^-6 to 10^7 /kb (per gene)

the Ames test makes ____

practical use of bacterial mutations to detect for potentially hazarduous chemicals

the Ames test looks for an increase in ___

mutations of bacteria in the presence of suspected mutagen

- a wide variety of chemicals have been screened for toxicity and carcinogenicity

mutagens

chemical, physical, or biological agents that increase mutation rates

(several classes of chemical mutagens exist)

a class of chemical mutagens: nucleotide base analogs

resemble nucleotides, directly incorporated into DNA, introduce mutatiosn during replication

they look like nucleotide, but they are not

chemical mutagens that induce ____

chemical modifications, for example, alkylating agents such as nitrosoguanidine

already in DNA, not thru the bases but react directly thru DNA bases -> altering structure

a class of chemical mutagens: intercalating agents

that insert between base pairs and cause frameshift mutations

- for example, ethidium bromide

deletions or insertions

some chemical, flat, can insert between DNA replication

two main categories of mutagenic electromagnetic radiations:

non-ionizing and ionizing

non-ionizing (i.e., UV radiation)

• purines and pyrimidines strongly absorb UV
• pyrimidine dimer is one effect of UV radiation

ionizing (i.e., x-rays, cosmic rays, and gamma rays)

• ionize water and produce free radicals
• free radicals damage macromolecules in the cell

there ___ types of DNA repair systems

3

direct reversal (type of DNA repair system)

mutated base is still recognizable and can be repaired without referring to other strand (ligase)

enzyme can undo the damage restoring the residue base without needing to cut the DNA backbone or use the template

repair of single-strand damage (type of DNA repair system)

damaged DNA is removed and repaired of using opposite strand as template

basic incision repair

nucleotide incision repair

repair of double-strand damage (type of DNA repair system)

a break in the DNA

can lead to chromosome fragmentation

• requires more error-prone repair mechanisms
• homologous recombination - sister chromosome is available as a template
• or non homologous and joining - basically try to stick the broken ends together

perfect fidelity in organisms is counterproductive because ____

it prevent evolution

the mutation rate of an organism is subject to ___

change

mutant can be isolated from strains that are ____

hyperaccurate or hypermutable

mutator (hypermutable) strains:

bacteria that benefit from increased mutation rates, such as dnaQ mutants (dnaQ encodes the proof-reading enzyme in DNA polymerase III) - the enzyme that copies DNA, if dnaQ is mutated, the dna polymerase makes. more mistakes and mutation rate of the cell increases

SOS regulatory system ___

is more error-prone. it allows replication to proceed and cell to replicate, but errors are more likely

carried out by DNA polymerases IV and V

SOS system allows DNA to be synthesized with no template

when DNA damage is large scale, the cell may use a different type of repair system (i.e., damage interferes with DNA replication)

image:

1. RecA gets activates and then acts like a coprotease

2. which activates lexA to cleave itself (to be destroyed), its a repressor protein, that normally sits on the DNA and blocks the expression of genes of DNA repair (gets degraded)

3. break comes off, the gene that it was repressing get switched on

when lexA levels drop: (they form component of polymerase) they are low fidelity transleisiancy polymerases- just copy pass damaged dna polymerases, they guess or skip over the DNA damaged part

uvrA partial repression (part of more accurate nucleotide excision repair pathway)

umuCD full activation of genes

trading accuracy for survival

three mechanisms of genetic exchange:

transformation

transduction

conjunction

three possible fates of horizontal gene transfer

degradation

replication

recombination

gene transfer in bacteria - genetic recombination

recombination:

physical exchange of DNA between genetic elements

homologous recombination:

process that results in genetic exchange between homologous DNA from two different sources. recA is essential.

facilitating genetic variation and the repair of DNA damage

selective medium can be used to ____

detect rare genetic recombinants

a simplified version of homologous recombination (video)

transformation

genetic transfer process by which DNA is incorporated into a recipient cell and brings about genetic change

competent cells (recipient cell)

cells are capable of taking up DNA and being transformed

• many gram + are naturally competent
• in naturally trasnformable bacteria, competence is regulated
• in other strains, specific procedurers are necessary to make cells competent
• chemical (such as Ca+2) treatments can turn cells competent
• electricity can be used to force cells to take up DNA (electroporation) allows DNA to enter

natural transformation of gram + by single-strand DNA (photo)

transduction

transfer of DNA from one cell to another by a bacteriophage

two modes: generalized transduction and specialized transduction

generalized transduction

DNA derived from virtually any portion of the host genome is packaged inside the mature virion

• defective virus particle incorporates fragment of the cell's chromosome randomly
• low efficiency

specialized transduction

DNA from a specific region of the host chromosome is integrated directly in the virus genome

• DNA of temperate virus excises incorrectly and takes adjacent host genes along with it
• transducing efficiency can be high

phage conversion

alteration of the phenotype of a host cell by a lysogenization (phage DNA incorporates into bacterial chromosome and becomes dormant)

• nondefective temperate phage lysogenizes a cell and becomes a prophage
• host cell becomes immune to further infection by same phage
• other phenotypic changes can also occur
• - Salmonella enterica serovar Anatum and bateriophage e
• - Corynebacterium diphtheriae and bacteriophage B

bacterial conjugation (mating)

mechanism of genetic transfer that involces cell-to-cell contact

• plasmid-encoded mechanism
• donor cell: containsconjugative plasmid (F+)
• recipient cell: does not contain plasmid (F-)

F(fertility) plasmid

• circular DNA molecule; ~100 kbp
• contains genes that regulate DNA replication
• contains several transposable elements that allow the plasmid to integrate into the host chromosome
• contains tra genes that encode transfer functions

DNA synthesis is necessary for ___

DNA transfer by conjugation

-DNA synthesized by rolling circle replication

images of F-pilus

transfer of plasmid DNA by conjugation (photo)

F plamids can integrate into ___

host chromosome

cells possesing a nonintegrated F plasmid are called ___

F+

cells possessing an integrated F plasmid are called ___

Hfr (high frequency of recombination)

• high rates of genetic recombination between genes on the donor chromosome and those of the recipient

presence of the F plasmid results in ____

alterations in cell properties

• ability to synthesize F pilus
• mobilization of DNA for transfer to another cell
• alteration of surface receptors so that cell can no longer act as a recipient in conjugation

insertion sequences (mobile elements) are ___

present in both F plasmid and E coli chromosome, which facilitate homologous recombination

plasmid is now part of chromosome. chromosomal genes transferred with plasmid

the formation of an Hfr strain (photo)

recipient cell remains as F- and does NOT become Hfr because ____

only a portion of the integrated F plasmid is transferred by the donor

discrete segemnts of DNA that move as a unit from one location to another within other DNA molecules are _____

transposable elements

transposable elements can be found in all ___

three domains of life

move by a process called transposition

• frequency of transposition is 1 in 1,000 to 1 in 10,000,000 per generation
• first observed by Barbara McClintock

transposable elements: Two main types of transposable elements in Bacteria are ____ and ____.

transposons and insertions sequences

• both carry genes encoding transposase
• both have inverted repeats at their ends

insertion sequences

are the simplest transposable element

• ~1,000 nucelotides long
• inverted repeaats are 10-50 base pairs
• only gene is for the transposase
• found in plasmids and chromosomes of Bacteria and Archaea
• found in some bacteriophages

only encode one protein

transposons

are larger than insertion sequences

• transposase moves any DNA between inverted repeats
• insertion of a transposable element generates a duplicate target sequence
• may include antibiotic resistance

- ex. Tn5 (kanr) and Tn10 (tetr)

often times carry useful genes

major player in the spread of antibiotic resistancy

transposition replicates target sequence (photo)

using transposons to make mutants:

• Transposons with antibiotic resistance are used
• Transposon is on a plasmid that cannot be replicated in the cell
• Cells capable of growing on selective medium likely acquired transposon
• Most insertions will be in genes that encode proteins
• Next step: screen for loss of function to determine insertion site

Preserving Genome Integrity: CRISPR Interference

CRISPR: ____

Clustered Regulatory Interspaced Short Palindromic Repeats

Type of prokaryotic "immune system"

• Region of bacterial chromosome containing DNA sequences similar to foreign DNA (spacers) alternating with identical repeated sequences

• CRISPR-associated proteins (Cas proteins)

• Obtain and store segments of foreign DNA as spacers • Recognize and destroy foreign DNA
• protect cell from repeat infections

Genome Editing and CRISPRs

• Sequence targeting by the Cas9 protein

• Cas proteins of CRISPR systems function as endonucleases when guided to nucleic acids.
• Synthetic RNA (synthetic guide RNA [sgRNA]) that recruits Streptococcus Cas9 and binds to target DNA enables cutting in genome of almost any cell; DNA can be ligated or used to insert new DNA.
• Cutting requires protospacer adjacent motif (PAM, 3 bp)
• Homologous recombination can be used to incorporate new DNA.
• Nonhomologous double-stranded DNA break repair pathway can ligate after deletion.

Operation of the CRISPR system (photo)

CRISPRs and genome editing (photo)

The applications of CRISPR-Cas9 (photo)