chapter 14: site specific recombination-- exam 2 Flashcards
what is site specific recombination?
rearrangement between 2 specific DNA sequences
What does site specific combination result in?
- insertion
- deletion
- inversion
Site specific recombination requires ______ and ______
- restriction endonucleases
and - ligases
T/f Site specific recombination is isoenergenic and doesn’t require extensive homology
true
What is the earliest form of parasitism?
Transposition
What is transposition?
When transposons move freely between different chromosomes
Where are recombination sites located?
at inverted repeats with a non palindromic core
Does the non palindromic core have direction?
yes
Describe the direction of the non palindromic core
- recombination with cores in opposite directions lead to inversions
- recombination with cores in the same directions lead to deletions
- between 2 separate chromosomes, same direction cores lead to insertions
recombination with cores in opposite directions lead to ______
inversions
recombination with cores in the same directions lead to _______
deletions
between __________, same direction cores lead to ______
2 separate chromosomes ; insertions
Active sites are typically ___ or ___ residues as nucleophile
Ser ; Tyr
_ light gray recombinases are active at a time
2
site specific reaction is very similar to the process of _____
topoisomerases
What are some examples of site-specific recombination?
- plasmid replication
- viral DNA circularization
- biotech
- phase variation
What are bacteriophages?
viruses that infect bacteria
What are the 2 pathways of viral infections?
- lysogenic pathway
2. lytic pathway
LoxP is…
site specific recombination loci
- allows for circulation of bacteriophage DNA
What is Salmonella typhimurium?
Regulates gene expression via site-specific recombination
•Flagella is a prominent target of mammalian immune systems
•Can change protein types of flagella to avoid host immunity!
•FljB FliC
Pieces of DNA that move from one part of the genome to the other (donor –> target site)
- can be random or specific
Transposons
What are the 3 types of transposons?
- cut and paste
- replicative
- RNA intermediate (retrotransposon)
________ is coded for within the transposable element transposon
transposase
What 2 things does transposes catalyze?
- hydrolytic cleavage
2. Transesterification
What is a cut and paste transposition?
•Double-stranded cuts in donor DNA—must be repaired*
•Liberated DNA attacks target DNA via 3’ –OH groups to form new phosphodiester bonds
•Single-stranded gaps filled in with repetitive DNA
*Donor DNA can be repaired through ligation or recombination repair
What are the 2 types of retrotransposons?
- Long-terminal repeat with extrachromosomal primed retrotransposition
(LTR (EP)) - Non-long-terminal repeat with target-primed retrotransposition
(Non LTR (TP))
LTR (EP) Retrotransposons:
- Transposon with a long-terminal repeat and extrachromosomally primed retrotransposition
- Long-terminal repeat = repetitive DNA at the termini of transposon between 100 – 5000 bp
- Extrachromsomally primed = DNA primers come from tRNA (not part of the chromosome)
Mechanism of LTR (EP) Retrotransposons:
- Transposon is transcribed by RNA polymerase ssRNA transcript
- Reverse transcriptase ds-cDNA
* •Remember, DNA always requires a primer! In this case . . . tRNA . . . wild! - Circularized by integrase (related to transposase)
- Invasion of target DNA
- Donor and target both have copy of transposon
* Reverse transcriptase can make dsDNA out of ssRNA. That is, it can use DNA and RNA as a template.
Non-LTR (TP) Retrotransposons
- Transposon without long-terminal repeat, with target-primed retrotransposition
- Uses part of the ssRNA intermediate as a primer for DNA
Bacteria have 3 common classes of transposons, what are they?
- Insertion sequences
- Composite transposons
- Complex transposons
Generally, do bacterial transposons have RNA intermediates?
NO
What do Insertion sequences contain?
Only DNA sequences necessary for transposition
What do composite transposons contain?
DNA sequences necessary for transposition and other genes
Complex transposons have:
- large genome
- genes for other processes than transposition
- act as transposon or bacteriophage DNA
(lytic and lysogenic pathway_
What percent of human DNA in each cell consists of transposons?
46%
What percent of transposons in human DNA come from retrotransposons?
90%
What percent of humans retrotransposons in their DNA are LTR vs Non-LTR?
LTR: 20%
Non- LTR: 80%
Describe LTR retrotransposons:
- Similar to viruses
- Transcribed with a poly-A tail•Transported to cytoplasm
- Translated to
- Gag = structural; creation of VLP
- Pol = reverse transcriptase
- Int = integrase
- Reverse transcribed and brought into the nucleus via VLP
- dsDNA integrates in new location
What are the 2 types of Non-LTR transposons?
LINEs and SINEs
What does LINEs stand for?
Long interspersed nuclear elements
What does SINEs stand for?
Short interspersed nuclear elements
Are LINEs autonomous?
Yes
Are SINEs autonomous?
No
Describe LINEs:
- Transcribed to RNA in nucleus
- RNA translated into cytoplasm
- Reintegrates DNA into the nucleus
- Autonomous
Describe SINEs:
•Transcribed to RNA in nucleus
•RNA translated into cytoplasm
•Reintegrates DNA into the nucleus
- Non-autonomous
What is the difference between SINEs and LINEs?
SINEs are non-autonomous
What are retroviruses?
Viruses that contain reverse transcriptase
What are the typical genes of retroviruses?
Gag = structural Pol = reverse transcriptase, integrase, proteases Env= structural ; allows element to transfer from cell to cell
What does viral reverse transcriptase lack?
3’ –> 5’ exonuclease activity
HIV is a retrovirus that infects ____ cells
T
What are the drug targets for HIV
- reverse transcriptase
- HIV proteases
Why is combination effective in fighting HIV?
because there is a very high mutation rate
