Lecture 15 Flashcards
What is the difference between wild-type and mutant?
wild-type organisms have the original DNA sequence and mutants have an altered DNA sequence
What is a transition mutation?
substitution of one pyrimidine for another or one purine for another in a base pair. ex) change of AT to GC
What is a transversion mutation?
substitution of a purine for a pyrimidine and a pyrimidine for a purine in a base pair. ex) AT to CG
What is a silent mutation?
codes for the same aa
What is a missense mutation?
encodes for a different aa
What is a nonsense mutation?
encodes for a stop aa
What is a frameshift mutation?
gain or loss of one or several base pairs so that the frame of codons is disrupted
What is an auxotroph?
requires an exogenous building block or growth factor (aa or vitamin)
Frameshift mutations almost always result in a loss of function?
yes
Microfluids
parent bacteria at top of tube and media runs through tube and washes progeny which is then independent of adapation
How do xrays and gamma rays affect DNA?
can cause double-strand breaks in DNA, the repair of which leads to macrolesions
How does UV light affect DNA?
cause adjacent pyrimidines in DNA to join at positions 4 and 5, forming dimers. Repair of the dimers results mostly in transversions, but also in frameshifts and transitions
What are 2 chemical agents of mutations?
Base analogs or DNA modifiers
What is a base analog?
Incorporated in DNA instead of dNTP
* Can pair with incorrect nucleotide during replication and cause mutation
What is a DNA modifier?
Deamination by nitrous acid converts cytosine to uracil
* During next round of replication uracil “read” as a thymine and base-paired with adenine (CG-to-TA transition
Why did penicillin enrich for His- mutants in the previous experiment?
Penicillin only kills dividing cells
What are transposons?
all organisms carry this, carry a segment of DNA to another locations….”jumping genes”
all contain inverted repeats
What catalyzes transposons?
transposase enzyme
What is a insertion sequence (IS) element?
doesn’t do much
What can encode drug resistance?
non-composite transposons —- carry whole gene
What are composite transposons?
contain two IS elements flanking additional genes
Making directed mutations requires which of the following?
homologous recombinations
What are polar effects?
Since prokaryotic genes are mostly in operons, gene deletions or disruptions can have “polar affects” where downstream genes are also affected and one of these genes may actually be responsible for the phenotype
What is complementation?
Add back a good copy of the gene and see if the phenotype reverts to wild-type
What is transformation?
naked DNA (like mailing a postcard)
What is transduction?
DNA within a phage (like mailing a letter in an envelope)
What is conjugation?
Direct transfer (handing someone a birthday present)
Collectively known as Horizontal Gene Transfer (HGT)
What does competent mean in fransformation?
Cells capable of taking up DNA from their environment
How does natural transformation work?
- Exogenous double-stranded DNA binds to proteins of the competence system located on the cell membrane
- The DNA is fragmented into smaller pieces
- One strand is degraded by a nuclease while the other enters the cell
- As the strand enters the cytoplasm it is coated with the RecA protein that protects it from exonucleases in the cytoplasm
- If part of the DNA is homologous to the cell’s resident DNA, RecA facilitates its integration into the chromosome by homologous recombination
Transformation only occurs in cells that are?
competent
What are 2 methods of artificial transformation?
chemical = heat shocked
electroporation = tiny holes are created by high-voltage jolts
How does conjugation in Gram-negative bacteria?
plasmids transferred through a conjugative pilus and T4SS, F+ transfers to F-
What is Hfr?
high frequency of integration, when plasmid from conjugation is incorporated into host genome
How does conjugation in Gram-positive bacteria?
plasmids is mediated by pheromones and not a pilus,
- The recipient cell produces chromosomally encoded pheromone cA
- cA interacts with plasmid pA in the donor cell causing it to produce aggregation substance (AS)
- AS binds to binding substance (BS) on other cells thereby clumping them
- IcA inhibits cA production in donor cells ensuring that clumping only occurs with recipient cells
- Once clumped, the plasmid is transferred
Which mediates transduction?
phages
