Recombination Flashcards
Recombination - what is it & what does it include
-why it’s important
- is the cutting and joining of DNA
- crossing over at meiosis is also classed as recombination
- Is the real driver of genetic variation
- allows much greater genetic diversity = greater adaptability to changing conditions
Homologous sequence that recombination can occur
- Crossing over or recombination occurs between homologous sequences
- Homology = 2 sequences that are similar along their length, but not necessarily identical
- can undergo exchange of DNA which can lead to a chimeric molecule (something that didn’t exist before)
How does crossing over occur? OVERVIEW
- Sister chromosomes align themselves at regions of homolgy
- need ss cuts in DNA so strands can separate and recombine to form a heteroduplex DNA
- is then repaired by mismatch repair system to give recombination products
Crossing over in homologous chromosomes
-proteins involved and what happens
- Initial alignment is mediated by the recA protein
- recA forms filament on DNA (has ss & ds inputs - changes them around in output)
- strand exchange reaction occurs (due to recA)
- Single stranded breaks are made by the recBCD complex (3 proteins)
- anything that induces ss or ds breaks WILL stimulate recombination
- recA is also involved in pairing and strand exchange reaction
- once strand exchange occurs, the cross over junction migrates to extend the heteroduplex (mediated by ruv AB complex)
Defects in rec proteins - what do they do
-why mutations in rec A is much worse than recBC mutants
- defects in rec proteins increase the sensitivity to DNA damaging agents
- rec A mutant is much more sensitive than recBC mutants
- if no recA, can’t repair lesions by combination OR induce genes for SOS repair system
RuvAB complex - role
- Extends regions of heteroduplex
- after migration of the junction, the junction is cut to release the products
What happens when there is a mismatch in DNA due to recombination
-how to know which strand is ‘right’
- The ‘right’ combination could be either of the two strands. (as both strands will be methylated)
- The mismatch repair system repairs the DNA but could give EITHER of the two genotypes
- frequency at which genotype is recovered depends on the bias of the mismatch repair (not always 50-50)
- bias depends on the location of the mismatch (tho not fully understood why)
- frequency at which genotype is recovered depends on the bias of the mismatch repair (not always 50-50)
- in some cases, the mismatch repair system will be completely in favour of one genotype = gene conversion (that gene would completely disappear)
Illegitimate Combination
- intramolecular recombination inversion
- inversion
- deletion
-Is combination that occurs outside cell division
-Intramolecular recombination inversion can occur with mobile genetic elements (they move and leave a copy and copies can recombine)
Inversion: moves gene - has significant effect on gene expression (as location on chromosome plays a big part)
-heterochromatin and euchromatin
-deletion: if sites are directly repeated, can cause deletion
Translocation of chromosomes (Illegitimate combination)
- leads to trisomies
- in plants, an extra chromosome can be tolerated, in animals, extra chromosome isn’t dealt with as well.
3 general recombination proteins
- recA
- recBCD
- ruvA-ruvB
*called general because they mediate recombination between any homologous sequences
Site Specific Recombination
- what it is
- most well known
- Occurs between specific sequences and is mediated by specific proteins
- best understood of these = integration of lamda phage into the genome
- insertion of phage to bacterial genome occurs at specific place
- best understood of these = integration of lamda phage into the genome
2 things required for Site specific recombination
- Integrase: Important enzymes that cuts the aHP and aHB sites and then joins the lambda DNA to the end of a chromosome
- responsible for bringing together the interacting components (this is a lambda protein)
- IHF (integration Host factor
Process of excising lambda DNA from site specific recombination
-what it requires
- When cell gets damaged, gets non coding lesions in DNA which induces the synthesis of Sos genes
- get increased level of beta genesis due to presence of Error prone DNA poly
- recA protease cleaves lexA repressor and C1 repressor of lambda prophase -> causes excision of chromosome and causes it to go through lytic cycle
- Sos induction will reverse reaction to excise lambda from the chromosome
- requires integrase, lambda XIS enzyme and IHF
Transposons and Insertion sequences
- are sequences that can move around the genome from one place to another
- Insertion sequences are usually 1kbp in length
- characteristics of these elements = inverted repeats at each end
- between repeats = gene for transposase
Complex transposons in bacteria
- e.g. Tn10 which is about 10kbp
- DNA is flanked by 2 copies of IS10 (insertion sequences)
- 1 of the IS10 contains a defective transposase gene, other one contains functional transposase gene
- tetracycline resistance carried between IS10
- a lot of transposons carry antibiotic resistance genes
- whole unit capable of migration as a unit
Important property of IS10
- transposon also makes ‘RNA out’
- out and transposase mRNA are complimentary, but overlap at the 5’ ends
- the 2 RNAs anneal which prevents translation of the transposase mRNA
- out and transposase mRNA are complimentary, but overlap at the 5’ ends
- RNA out always present in excess, means annealing will almost always take place
What sequences such as Tn10 can do
- sequences such as Tn10 provide a substrate for general recombination proteins -> can lead to deletions, inversions, etc.
- can also get translocations occuring
- Genome evolution can be induced/stimulated by mobile genetic elements
Complex transposons -> habit
- like to cluster where other transposons are
- Tn5385 is approx. 65kbp
- whole thing can move, or segments within it can move
- carries genes that mostly contain antibiotic resistance that can jump into plasmids
- whole thing can move, or segments within it can move
- Because mobile elements insert into regions where there is already Tns, arrays of resistance genes are built
Retroelements
- what they are
- important enzyme
- retroelements in our genome
- These transfer via an RNA intermediate
- reverse transcriptase is responsible for this (is found in retro DNA -> it mediates the copying and integration reaction
- lot of retroelements in our genome -> most of our cells will have reverse transcriptase activity
mRNA -> cDNA -> inserts into genome -> psuedogene
Non-autonomous elements
MITEs
- Non-autonomous elements: lost their transposase - therefore rely on it coming from another source
- MITES: Minimal inverted transposable elements
- dont have their own genes (can insert into gene which disrupts the gene or can insert into a promoter and interact with it)
- MITEs can therefore have biological activity even though it doesn’t have genes
Genomic Islands - what they are and what they do
- When looking at closely related bacterial species, in certain regions there aren’t homologous sequences -> usu in blocks
- differences aren’t random - occur in discrete areas called genomic islands
- Genomic islands can transfer b/w species and variants of the same islands can be found in related species
Functions of genomic islands
Functions include;
- Degradation of pehoic compounds
- Iron uptake
- Pathogenicity factors
- Nitrogen fixation
- DNA secretion system
- multiple antibiotic resistance
Horizontal Gene transfer (HGT) - what is it, why important
- 2 ways it can be detected by
- Transduction - what is it and what systems bacteria have to regulate DNA
*is genetic transfer from other species
-is important for gaining new genetic info for a species
Can be detected by;
-difference in % GC
-difference in codon preference
-Transduction: Where a bacteriaphage picks up DNA from one bacteria and transfers it to antoher
-bacteria have a restriction/modication system to ensure that only DNA from the same species is taken up and incorporated
-other DNA will have a different modification site and will be degraded
DNA transfer from bacteria to eukaryotes
e.g. of what can do it (and what it does)
- e.g. agrobacterium Iumefaciens (is a plant bacterial species)
- infects wound sites, only strains with tumor inducing plamid
- It cuts out a section of this plasmid and transfers it to the DNA of the plant (called T-DNA)
- relatives of this can do the same thing
- e.g. gus gene = when expressed gives a blue product (designed to be expressed in plan, but good indicator of transfer)
Fungi-Bacteria transfer
- Some fungi infect plants and get into cells and tissues - high potential for DNA transfer
- more we sequence, more we can see it happening
- also see plants to fungi DNA transfer
DNA transfer between plant species
- Particularly happens through interspecific hybridization (has been used by breeders to breed new varieties of flowers)
- plants quite flexible as to what you do with the genome
Animal to animal gene transfer
- Animals are more reproductively isolated and are less amenable and receptive to interspecific hybridization
- insects can cause the transfer of DNA b/w species (esp. blood sucking insects)
- diet is another way DNA can be transferred
Frequency of integration
- why is what it is
- exception
-is very low (approx 10^-3) (for every 1000 that integrate into cell, only 1 will integrate into the genome)
UNLESS the DNA encodes its own integration enzyme
-mobile genetic elements do this, whereas normal DNA doesn’t
-DNA needs to get into genome (not just into the cell)
Consequences of transfer of MGE?
- Provide regions of homology
- Stimulate DNA rearrangement
- provide gene regulatory sites (can provide new cis-regulating sequences and can switch off genes)
Why do MGE prefer to integrate adjacent to genes that are expressed (2 reasons)
- Strands of DNA are separated (therefore easier to invade)
2. Genes expressed in euchromatin (aren’t as densely packed as those unexpressed genes in heterochromatin)
Post transcriptional effects of MGE
- Provide extra exons
- effect alternate splicing
- effect RNA stability
- Provide different site for polyadenylation
How DNA can be uptaken through diet
- DNA can survive and be absorbed through the wall of intestine and then circulate through blood
- Experiment using beta-galactosidase showed that this can occur in spermatozoa (which has low conc. of nucleases and more retrotransposases)
Endosymbiotic bacteria & DNA transfer
- Where bacteria has a long term relationship with its hos
- can’t live independently of host due to very small genomes
- Is a number of examples of when the cell uses endosymbiotic DNA in new ways
