Ex3 Transposons and site-specific recombination Flashcards
What are transposons?
Genes that can move (jump) from one DNA molecule to another in a cell or from one location to another on the same DNA molecule
What happens in the process called Transposition?
Genes that can move around to different positions within the genome of a single cell
What are “jumping genes”?
Transposons
What are the two ways of presenting Transposons?
Junk DNA - no known useful function
Selfish DNA- exploit the genetic mechanisms of the cell
What can transposons facilitate by transferring of genes in bacterial plasmids?
Antibiotic-resistance and can also contribute to mutations.
True or False: Transposons exist and the adjustability of genes or groups of genes is more important than the change of single genes (point mutations).
True
What are the 2 classes of Transposons?
Class I: Retrotransposons
Class II: Transposons
How do Class I transposons work?
By copying themselves and pasting copies back into the genome, in multiple places.
What are the 2 steps of Retrotransposons?
Copy themselves to RNA (transcription) but instead of being translated the RNA is copied into DNA by a reverse transcriptase and inserted back into the genome.
What can retrotransposons behave similarly to? (virus)
Retroviruses (HIV)
What do Class II transposons do?
Move directly from one position to another within the genome using a transposase to “cut and paste” them within the genome
True or False: Different types of transposase work in different ways. Some can bind to any part of the DNA molecule, and then target site can therefore be anywhere, while others bind to specific sequences. The transposase then cuts the target site to produce sticky ends, cuts out the transposon and ligases it into the target site, then fills in the sticky ends with their complementary base pairs.
True
If both classes of transposons lose their ability to synthesize reverse transcriptase or transposase through mutation, what can they do?
They can continue to jump through the genome because other transposons are still producing the necessary enzyme.
True or False: Some transposons can move directly from one position to another within the genome, while retrotransposons have to first be transcribed to RNA and then back to DNA by reverse transcriptase.
False; All transposons can move from one position to another within the genome.
How are transposons useful to researchers?
They act as a means to alter DNA inside a living organism
_______ is from where the transposon originated from. _______ is where the transposon hops to.
Donor DNA; Target DNA
What are the 3 general groups of bacterial transposons?
- Insertion sequence elements
- Composite transposons
- Non-composite transposons
___________, which only encode gene products required for transposase synthesis and regulation.
Insertion sequence elements
___________, is composed of any gene sandwiched between two IS sequences; this entire unit will move.
Composite transposons
__________, genes and the inverted-repeat ends are included in the minimum transposable element and are not part of autonomous IS elements.
Non-composite transposons
What is the simplest type of transposon?
Insertion sequence (IS)
What does an insertion sequence do and what is it?
IS is a sequence of DNA that encodes an enzyme called transposase, which allows the IS to move.
The transposase gene is flanked on either side by ______ to_____ base pairs, arranged as “inerted repeats”.
15 to 25 bp
What are the four IS elements?
IS1, IS2, IS3, and IS4
Plasmids also carries IS elements. Why is this important?
It is important in the formation of Hfr strains
What are the elements of an IS?
- All bacterial transposons contain repeats at their ends.
- The target DNA has a short direct repeat that bracket the transposon
- ORFA, regulate the transcription of the transposase
- ORFB, active transposase
What are composite transposons?
They are larger transposons formed for two IS elements bracketing other genes.
___________, each IS element can transpose independently as long as the transposase acts on both of its ends.
Out-site end transposition
How does composite transposons work?
The transposase acts in the distant IS ends. Then 2 IS elements along with genes in between will be transpose as a unit.
The transposase encode by one of IS elements in the ______ acts in IS elements that are closest to each other.
Composite
What are the consequences of inside-end transposition
- Creation of a new composite transposon.
- Deletion and inversion to a nearby target on the same DNA
Insite-end IS __________ cross over each other before attaching. Insite-end IS cross over ________ before attaching.
do not; each other
What are the origin of many plasmids?
The assembly of plasmids by IS element
What is the difference between an insertion sequence and a composite transposon?
The insertion sequence only contains genes for the mobility of the sequence; a transposon carries some other trait (such as antibiotic resistance) as well
What are the three important facts about non-composite transposons?
- Genes that are integral part of the transposon
- Resistance gene is part of the minimum transposable unit
- Resistant genes integrate into the transposon by specific integrase enzymes (lisogenic phage into the chromosome)
What are the properties of transposable genetic elements?
- Movement is not totally random
- Not capable of self replication
- Transposition mediated by site-specific recombination
- Transposition can be accompanied by duplication
True or false: Transposable genetic elements can move from any DNA molecule to any other DNA molecule or even to another location on the same molecule. The movement is not totally random; there are preferred sites in a DNA molecule at which the transposable genetic element will insert.
True
True or False: The transposable genetic elements exist autonomously ( except some transposable phages) and thus, to be replicated they must be a part of some other replicon.
False; Transposable genetic elements do not exist autonomously.
True or False: Transposition requires little or no homology between the current location and the new site. The transposition event is mediated by a transposase coded by the transposable genetic element.
True
Recombination that does not require homology between the recombining molecules is called ______ or ________ or ________.
Site-specific; illegitimate; nonhomologous recombination
True or False: In many instances transposition of the transposable genetic element results in removal of the element from the original site and insertion at a new site. However, in some cases the transposition event is accompanied by the duplicaitonn of the transposable genetic element. One copy remains at the original site and the other is transposed to the new site.
True
_______________, the entire transposon replicates during transposition resulting in two copies of the transposon.
Replicative mechanism
____________, the transposon is removed from one place and inserted into another (e.g. IS elements and composite transposons such as Tn10,ent)
Cut-and-Paste or conservative or non-replicative mechanism
_________, wherever the transposon hops into a site a short sequence of the target DNA is duplicated.
Replicative transposition
Formation of the _______, the donor and target DNA have become fused (encode 2 copies of the transposon)
Cointegrate
Donor and target DNA separate by the host recombination functions or by a transposon-encode_______ that promotes recombination at internal res sequences
resolvase
After resolution of the ________, the donor and target DNA molecules have a copy of the transposon.
Cointegrate
What is the first step to replicative transposition?
Transposase makes a single strand break at each junction between the transposon and the donor DNA (transposon-donor DNA one 5’ end and one 3’ end at each junction)
What is the second step to replicative transposition?
A double-stranded break in the target DNA (staggered), the nicks are separated by the same number of base pairs (Target DNA two 5’ ends and two 3’ ends)
What is the third step to replicative transposition?
5’ ends of the target DNA ligated to the 3’ ends of the transposon
What is the fourth step to replicative transposition?
The 3’ ends of the target DNA are used as a primer for replication (Replication proceeds over the transposon in both directions)
What is the fifth step to replicative transposition?
The new 3’ ends of the donor DNA ligate to the remaining 5’ ends of the donor DNA (Cointegrate)
What is the sixth step to replicative transposition?
Recombination at res resolves cointegrate
What are three important points on replicative transposition?
- Cointegrates are obligate intermediaries in replicative transposition
- After transposition a short target DNA sequences of define length is duplicated at each end of the transposon (replication proceeds from the straggered breaks over the transposon)
- It is independent of the host function, including DNA ligase ( transposase –> cuts and promotes ligation; resolvase –> promotes recombination)
What are the four steps of cut-and-paste transposition?
- Transposase makes a double-stranded break at the ends of the transposon
- Cut out the donor DNA
- Paste it into the target DNA at the site of the staggered break
- The single-stranded gaps created by the icks in the target DNA are filled in.
What are 3 main points for cut-and-paste transposition?
- No cointegrate intermediate
- Both strand of the DNA transpose
- Transposon leaves the donor DNA
