Lectures 28-30 / Chapters 11-12

Classes of mutations

• Point mutation/base substitution

• Insertion/deletion

• Inversion

Point mutation/base substitution

A single nucleotide is changed in a DNA sequence

Insertion/deletion

Involves the addition or subtraction of one or more nucleotides

What does an insertion/deletion mutation cause?

Frameshift (which is why its also know as a frameshift mutation). The frameshift causes all of the amino acids after the insertion or deletion to be abnormal.

Inversion

A fragment of DNA is flipped in orientation in relation to the DNA on the other side

Types of point mutations/base substitutions

• Silent mutation

• Missense mutation

• Nonsense mutation

Silent mutation

Change in nucleotide sequence that does not affect amino acid sequence, therefore has no effect on the protein synthesized.

*This is because there are multiple codons that can code for the same amino aid.

Missense mutation

Change in nucleotide sequence that does affect the amino acid sequence, therefore has an effect on the protein synthesized. Can be harmless, harmful, or beneficial.

Nonsense mutation

Change in nucleotide sequence results in a stop codon, which results in a short, non-functional protein.

What causes mutations? (3)

• A mistake my DNA polymerase that fails to be repaired

• Physical agents (cosmic rays, x-ray, UV radiation)

• Chemical agents (ROS)

Oxidation of nucleotides by ROS creates a…

mutagen

What do x-rays do to DNA?

DNA breakages

When this happens, anything can be inserted to fill the gap (mutation)

What does ionizing radiation do to DNA?

UV light causes a kink or a bend, which causes thymines to bond together. AKA Thymine Dimerization

Types of DNA repair (4)

• Base excision

• Methyl mismatch

• SOS (save our ship)

• DNA recombination

Base excision repair

Recognizes a specific damages base and removes it from DNA backbone

Methyl mismatch repair

Requires recognition of the methylation pattern in DNA bases

SOS repair

Coordinated cellular response to damage that can introduce mutations in order to save the cell

*a last-resort, mutagenic repair system — better for survival than death, but potentially harmful long-term.

DNA recombination

The process of crossing over and exchange of 2 DNA helices

Levels of gene regulation (4)

• Changing the DNA sequence

• Control of transcription

• Translational control

• Post-translational control

Changing the DNA sequence

Some microbes change the DNA sequence to activate or disable a particular gene

Control of transcription

Transcription can be regulated by protein repressors, activators, and alternative sigma factors

Translational control

The regulation of how efficiently mRNA is translated into protein

Post-translational control

Control of proteins that are already made, like activating and deactivating the protein, or protein degradation

Operon

ON THE DNA. Set of genes controlled by the same promoter

Promoter region

Can think of it like a “switch.” A segment of DNA that is upstream from a particular gene. RNA polymerase binds here, so does inducers

Control region of the operon is made up of…

Promoter and operator

When a regulator binds to the promoter…

When a regulator binds to the operator…

Activates transcription

Represses transcription

Regulatory gene

NOT A PART OF THE OPERON. Activated independently of the genes in the operon

Polycystronic mRNA

mRNA that can encode multiple proteins because it contains multiple genes (typical of bacteria)

In the absence of an inducer…

the repressor binds to the operator, preventing transcription

In the presence of an inducer…

Inducer binds to the repressor, preventing it from binding to the operator on the operon. Transcription occurs

How do cells sense the intracellular environment?

Cell can turn on certain genes based on its need/environmental signal. Different regulatory proteins bind to specific compounds to determine its concentration

Example of regulatory proteins bind to specific compounds to determine its concentration

dtx, the diphtheria toxin

When there are high levels of iron inside the cell, transcription is repressed (iron acts as repressor)

Global regulators

Proteins that affect the expression of many different genes

Example; cAMP (cyclic AMP)

How do cells sense the extracellular environment?

Relies on a series of two-component phosphorylation relay series

Example: Sensor kinase PhoQ in salmonella senses Mg and pH outside of the cell

Vertical gene transfer

Transmission of genetic material from parent to progeny

Horizontal gene transfer

Common in prokaryotes, transmission of genetic material between organisms that are not parent and offspring

Includes transformation, transduction, and conjugation

Transformation

• Uptake of DNA fragments from the environment

  • Typical from dead/lysed cells

  • Recipient cell must be “competent” (able to take up DNA)

Transformation is carried out by…

specific protein complexes called transformasomes

Which bacterial genuses are considered naturally competent?

Streptococcus, bacillus, haemophilus, neisseria

Which bacterial genuses can be manipulated to be artificially competent?

E. coli and salmonella

Electroportation

A brief electrical impulse that “shoots” DNA across the membrane

Transduction

Transfer of DNA via a bacteriophage (a virus). Originally discovered in salmonella by Joshua Lederberg.

Bacteriophage is composed of…

a core nucleic acid covered by a protein coat

Why is transduction significant? (5)

1. Transfer genetic material from one bacterial cell to another alters genetic characteristics

2. Incorporation of phage DNA into bacteria chromosomes demonstrates close evolutionary relationship

3. The fact that a prophage can remain for large periods of time suggests a similar mechanism for the viral origin of cancer

4. The viruses bring along genes from their previous host (or hosts). If this virus infects us, we could be considered transgenic

5. Provides a way to study gene linkage via chromosome mapping

Conjugation

Direct transfer of DNA via a pilus, donor must have conjugated plasmid.

Which allows for the largest quantity of DNA to be transferred?

1. Transformation

2. Transduction

3. Conjugation

Conjugation

Conjugation pathway

1. F+ donor cell attaches to recipient cell. Pilus contracts, drawing cells together to make contact

2. One strand of F plasmid DNA transfers from donor cell to recipient cell

3. Donor synthesizes complimentary strand to restore plasmid. Recipient creates complimentary strand to become F+ cell with pilus

Hfr cells

The F plasmid can occasionally integrate into the bacterial chromosome, producing an Hfr cell.

When there is an integrated F plasmid

Why is conjugation significant? (5)

1. Contributes to genetic variation

2. Increases genetic diversity because larger amounts of DNA are transferred

3. Represent evolutionary stage between asexual processes and the actual fusion of whole cell

4. Plasmids are self transmissable self-transmissible and can be promiscuous

5. Some Gram + bacteria have self-mating plasmids without F pili, they secrete peptide compounds to make bacteria that do have the plasmid mate with them

Plasmids

1. Mostly circular

2. Double stranded

3. Extrachromosomal

4. Self-replicating

5. Advantageous to bacterial cell

Plasmid functions

1. Encodes for sex pili protein

2. Resistance plasmids carry genes resistant to antimicrobials

3. Virulence plasmids cause disease signs and symptoms

4. Tumor inducing plasmids cause tumors in plants

5. Genes for catabolic enzymes

6. Bacteriocinogen plasmid

Transposable elements

Genes that can move from one chromosome to another

Can TEs replicate independently?

No

Transposase

Enzyme that moves the element from one gene to another

Restriction endonuclease

Enzyme that cuts pieces of DNA at a specific site

The restriction sequence in an endonuclease is a…

palindrome

Gel electrophoresis

Separate DNA fragments based on their size