Ch. 8 - Bacterial Genetics

Vertical Gene Transfer

1. one generation to the next

2. genes passed via replication to progeny

3. “parent to daughter cell”

Horizontal gene transfer

Friend to friend (same generation)

What are the 3 mechanisms of horizontal gene transfer?

1. Bacterial transformation

2. Conjugation

3. Transduction

Mutations

Changes to DNA sequence

Spontaneous mutation

When DNA polymerase makes a mutate in replication

What are two types of spontaneous mutations?

1. Base mutation

2. Frame shift mutation

Genotype

1. sequence of nucleotides in DNA

2. result of a change to an organism’s DNA

Phenotype

1. Organism’s observable characteristics

2. Result of change in genetic makeup or environmental influence

What are the 3 outcomes of base mutations?

1. Silent mutation

2. Missense mutation

3. Nonsense mutation

Silent mutation

1. Change generates a codon that translates into the same amino acid as the original

2. Can occur because of the redundancy in the genetic code (most amino acids are coded for by more than one codon - aka “wobble”)

3. Example:

   1. Original DNA: TGT —> ACA = UGC = Cystine

   2. Mutated DNA: TG(C) —> AC(G)

   3. mRNA: UGC

   4. Amino Acid: Cystine (for both)

Missense mutation

1. altered codon codes for a different amino acid

2. Example

   1. Original DNA: TGT —> ACA = UGC = Cystine

   2. Mutated DNA: TG(G) —> AC(C)

   3. mRNA: UGG

   4. Amino Acid: TRP (change in amino acid)

Nonsense mutation

1. base substitution creates a stop codon = shorter, likely non-functional protein

2. Example:

   1. Original DNA: TGT —> ACA = UGC = Cystine

   2. Mutated DNA: TG(A) —> AC(U)

   3. mRNA: UGA

   4. Amino Acid: Stop codon

Reading frame

1. grouping of a stretch of nucleotides into sequential triplets that code for amino acids

2. an mRNA molecule has 3 possible choices, but only one is typically used in translation

Met (M) or Start codon

AUG

Stop Codons

1. UAA

2. UAG

3. UGA

Frameshift mutation

nucleotide insertion or deletions that SHIFTS the reading frame

Why is a silent mutation not a possible result of a frameshift mutation?

A silent mutation does not alter the resultant amino acid and a frameshift mutation would irrevocably alter the sequence of base pairs through addition or deletion

What 2 mutation types are possible with a frameshift mutation?

1. Missense mutation

2. Nonsense Mutation

Induced mutations

Result from outside influence (not DNA polymerase mistakes)

Mutagen

agents that induce change

Alkylating Agents

1. modifies the bases

2. adds alkyl groups (-CH3 and others) to nucleobases

3. leads to incorrect base-pairing

4. Example:

   1. adds methyl to guanine making methylguanine

   2. mG sometimes pairs w/ T

Base Analogs

1. modifies the bases

2. resembles the bases, but have different hydrogen bonding properties

3. leads incorrect base-pairing

4. Example:

   1. 5-bromauracil (sub for T) - base pairs with A & C

   2. 2-aminopurine (aub for A) - base pairs with T & C

Intercalating agents

1. insert between adjacent base pairs in strand of DNA

2. distorts DNA shape

3. Leads to error in replication

4. Example:

   1. ethidium bromide

Radiation

1. UV light creates covalent bond between adjacent thymine molecules on strand of DNA (thymine dimer)

2. thymine dimer creates a “kink”

3. “kink” in DNA strand prevents correct-base pairing in next round of replication

4. Leads to error in replication

Proofreading

1. Repair of spontaneous mutations

   1. DNA Polymerase III goes back, catches mistake base-pairing & replaces with correct one

Mismatch repair

1. Repair of spontaneous mutations

2. Template DNA is methylated (-CH3)

   1. Nuclease removes section on methylated strand (newly made strand w/ mutation)

   2. DNA polymerase I: adds back correct nucleotide

   3. DNA Ligase: seals the gap

Excision Repair

1. Repair of induced mutations

2. Repair thymine dimers

   1. Nuclease removes T-T

   2. DNA polymerase: adds back nucleotides based on the template strand

   3. DNA ligase: seals the gap

Photoreactivation

1. Repair of induced mutations

2. Repairs thymine dimers with help of light

3. Light activates enzymes which breaks incorrect bond joining thymines

Direct selection

Cells inoculated onto medium that supports growth of mutant, but not parent (wild-type)

Auxotroph

1. Mutant

2. requires complex medium w/ all nutrients to grow OR minimal medium w/ required nutrient (i.e. arginine)

Prototroph

1. Typical wild-type parent-strain

2. Can grow on minimal medium that lacks added nutrients (i.e. can make their own arginine)

Indirect selection

To isolate an auxotrophic mutant through the use of complex medium vs minimal medium

Recombinants

acquire genes from other cells by horizontal gene transfer

True or False: Spontaneous mutations are a result of changes in DNA seqeunce due to the addition of a mutagen

False

A base substitution (point mutation) resulting in a premature stop codon is known as a ________ mutation

Nonsense

Direct selection

cells inoculated onto medium that supports growth of mutant (resistant), but not parent parent (wild-type)

Replica plating

Indirect selection of auxotrophic mutants; mutants are identified by comparing the growth of colonies on a nutrient agar plate and glucose-salts agar plate

DNA-mediated transformation

involves the uptake of “naked” DNA by recipient (competent) cells that is later integrated into the chromosome

What is a “competent” cell?

A cell that is able to take up DNA regardless of its source and integrate it into its chromosome

Transduction

Bacteriophages transfer bacterial genes from a donor to a recipient cell using a transducing particle

What error occurs that allows for generalized transduction?

A fragment of bacterial DNA - produced when the phage-encoded enzyme cuts the bacterial genome - mistakenly enters the phage protein coat

Through what mechanism can a transduced bacterial DNA integrate into a recipient’s chromosome?

Homologous recombination

What is a special feature of plasmids?

They are replicons - can be replicated without integrating into the recipient’s chromosome

What is the function of conjugative plasmids?

Direct their own transfer from donor to recipient cells

Have their own origin of replication & can replicate DNA in each cell division

F. plasmid