Recombination

Question 1

Recombination - what is it & what does it include

-why it’s important

Answer 1

• 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

Question 2

Homologous sequence that recombination can occur

Answer 2

• 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)

Question 3

How does crossing over occur? OVERVIEW

Answer 3

• 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

Question 4

Crossing over in homologous chromosomes

-proteins involved and what happens

Answer 4

• 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)

Question 5

Defects in rec proteins - what do they do

-why mutations in rec A is much worse than recBC mutants

Answer 5

• 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

Question 6

RuvAB complex - role

Answer 6

• Extends regions of heteroduplex

- after migration of the junction, the junction is cut to release the products

Question 7

What happens when there is a mismatch in DNA due to recombination

-how to know which strand is ‘right’

Answer 7

• 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)
• in some cases, the mismatch repair system will be completely in favour of one genotype = gene conversion (that gene would completely disappear)

Question 8

Illegitimate Combination

• intramolecular recombination inversion
• inversion
• deletion

Answer 8

-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

Question 9

Translocation of chromosomes (Illegitimate combination)

Answer 9

• leads to trisomies

- in plants, an extra chromosome can be tolerated, in animals, extra chromosome isn’t dealt with as well.

Question 10

3 general recombination proteins

Answer 10

• recA
• recBCD
• ruvA-ruvB

*called general because they mediate recombination between any homologous sequences

Question 11

Site Specific Recombination

• what it is
• most well known

Answer 11

• 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

Question 12

2 things required for Site specific recombination

Answer 12

• 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

Question 13

Process of excising lambda DNA from site specific recombination

-what it requires

Answer 13

• 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

Question 14

Transposons and Insertion sequences

Answer 14

• 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

Question 15

Complex transposons in bacteria

Answer 15

• 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

Question 16

Important property of IS10

Answer 16

• 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
• RNA out always present in excess, means annealing will almost always take place

Question 17

What sequences such as Tn10 can do

Answer 17

• 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

Question 18

Complex transposons -> habit

Answer 18

• 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
• Because mobile elements insert into regions where there is already Tns, arrays of resistance genes are built

Question 19

Retroelements

• what they are
• important enzyme
• retroelements in our genome

Answer 19

• 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

Question 20

Non-autonomous elements

MITEs

Answer 20

• 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

Question 21

Genomic Islands - what they are and what they do

Answer 21

• 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

Question 22

Functions of genomic islands

Answer 22

Functions include;

• Degradation of pehoic compounds
• Iron uptake
• Pathogenicity factors
• Nitrogen fixation
• DNA secretion system
• multiple antibiotic resistance

Question 23

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

Answer 23

*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

Question 24

DNA transfer from bacteria to eukaryotes

e.g. of what can do it (and what it does)

Answer 24

• 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)

Question 25

Fungi-Bacteria transfer

Answer 25

• 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

Question 26

DNA transfer between plant species

Answer 26

• 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

Question 27

Animal to animal gene transfer

Answer 27

• 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

Question 28

Frequency of integration

• why is what it is
• exception

Answer 28

-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)

Question 29

Consequences of transfer of MGE?

Answer 29

• Provide regions of homology
• Stimulate DNA rearrangement
• provide gene regulatory sites (can provide new cis-regulating sequences and can switch off genes)

Question 30

Why do MGE prefer to integrate adjacent to genes that are expressed (2 reasons)

Answer 30

1. 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)

Question 31

Post transcriptional effects of MGE

Answer 31

• Provide extra exons
• effect alternate splicing
• effect RNA stability
• Provide different site for polyadenylation

Question 32

How DNA can be uptaken through diet

Answer 32

• 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)

Question 33

Endosymbiotic bacteria & DNA transfer

Answer 33

• 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