MIC102 S9 - Mutation, Info Transfer

Explain how a change in genotype (mutation) contributes to a change in the phenotype

1) Mutation causes change in DNA sequence

2) Change in RNA and protein → diff AA, premature stop codon, etc.

3) Change in protein Shape/Function → enzymes, protein stability, interactions

4) Change in phenotypes

Describe strategies bacterial cells use to maintain a low mutation rate

1) High-Fidelity DNA Replication

• DNAP III 3’ → 5’ exonuclease activity

2) Post-Replication DNA Repair Systems

• Mismatch Repair

  • MutSLH system

3) DNA Methylation

• GATC hemi-methylation recognized by MutS

Other processes of cutting out DNA

Explain how different types of mutagens change DNA

Recombination

• mixing of genetic material

• occurs between regions with identical sequences

• facilitated by recombinases involved in DNA repair

• scale is variable; enitre genes or operons can be exchanged

• can result in a recombinant cell different from donor and recipient

Transposons

• Cut and paste transposition → involves Inverted Repeasts (IR) and Transposase

• Transposase binds to DNA sequence→ form transposition complex → excises transposon sequences → recognize target site and inserts to genome

Radiation

• Ionizing (x-rays, gamma) → cause single and double stranded breaks

• Ultraviolet (UV) → pyrimidine dimers (ex: T^T)

Others

• Chemicals

• mutator strains

List factors that determine whether a mutation has a large, small, or no effect on phenotype

1) When they occur

• Usually deleterious errors most costly to cell and progeny during DNA synthesis vs. trxn and trln → mess up blueprint and persist in future generations

2) Mutations types

• frameshfit and nonsense → reading frameshift and truncated proteins usually large effect

• silent mutation → no effect

• Missense → effect depends on importance of AA

3) Location of Mutation

• Exon → varying impact

• intron → no effect unless regualtory sequence

4) Function of Gene

Explain how the Griffith transformation experiment led to the discovery that phenotypes are determined by a transforming factor (DNA). Be able to interpret alternative outcomes from a similar experiment

Process

• Introduced 2 different strains of Streptococcus pneumoniae on mice

  • Rough: non-virulent (no capsule → no disease)

  • Smooth: virulent (has capsule → causes disease) -

  • Capsule helps prevent phagocytosis

• Focused on concept of transformation → cell takes up naked DNA and incorporates it to genome

Experimental conditions + interpretations

1) Live S strain

• Mouses dies

• Virulent Bacteria cause disease, Live S cells in blood

2) Live R strain

• Mouse lives

• Non-virulent, no capsule = no disease, no live R cells in blood

3) Heat killed S strain

• Mouse lives

• Bacteria dead = no disease, no live S cells in blood

4) Heal killed S cells + Live R cells

• Mouse lives

• R strain transformed into virulent S, Live R cells with S capsule in blood

Conclusions

• heat-resistant “transforming-porinciple” (DNA) form dead cells was acquired by the live cells from dead cells that could transform phenotype

• traits can be acquired and bacteria are good model to study genetics

Differentiate between transformation, transduction, and conjugation including key features of the donor and recipient (ex; live vs dead, required structures, etc.)

Transcormation

• uptake of naked DNA in environment by bacterial cells

• Donor → dead/lysed cell to release DNA fragments; Recipient → live, must be competnet to take up DNA

• Requires competence factors, DNA-bidning proteins on surface

Transduction

• Transfer of DNA via a bacteriophage (virus)

• Donor → Infected basterium and DNA gets mistakenly packaged into phage; Recipient → Live bacteria infected by phage

• Requires Bacteriophage to transfer

Conjugation

• direct DNA transfer via cell-to-cell contact

• Donor → live cell with conjugative plasmid (ex: F+ cell in E. coli); Recipient cell → Live cell lacking plasmid (F-)

• Required structures: sex pilus and conjugation machinery encoded by plasmid

List the steps in conjugation (for gram-negative bacteria) and transduction

Conjugation (Gram-Negative)

• Sex pilus forms between donor and recipient cell

• Sex pilus contracts and conjugation pore forms between cells for DNA repair

• Cut occurs at plasmid at origin of transfer, extends through pore

• Both cells use rolling circle replication to make complementary strand

• Both cells now are F+

Transduction

• Virion attaches and injects genome inside bacteria → encodes info to take over the bacteria

• Viral genome copies inside bacterial cell and does damage to bacterial genome

• Bacterial DNA is accidentally gets packaged inside a virion → Bacteria lyses and lets out virion into environment

• Virion with bacterial DNA attaches to recipient cells and injects genome

• Donor DNA and Recipient DNA recombine if homology present → transfer complete

Distinguish between natural and artificially-induced competence

Competence

• ability to actively take up free, foreign DNA; often transient and inducible

Natural Competence

• 1% of bacteria readily import DNA from their environment (in vitro) under permissive conditions

• inducible by high [cell], damage

Artificially-induced competence

• cation+heat or an electric pulse are used to make transient pores in the membrane