BIOL253 L10 and L11

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  • Created by: Katherine
  • Created on: 11-04-17 08:35
What is recombination?
Genetic exchange
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How do Eukaryotes achiece recombination?
Sexual cycle and meiosis
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How do prokaryotes achieve recombination as they divide by binary fission?
Conjugation, transformation, and transduction
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What do we know about E.coli genome?
Circular dsDNA chromosome defined as it's genome. Unique sentence. Contains extrachromosomal, small, circular DNA molecules (plasmids) which confer advantages.
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What are the 3 types of plasmid?
Sex plasmids, R plasmids, Col plasmids
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What is a sex plasmid?
It is the fertility plasmid of E.coli. It is is self mobile. It mediates transfer of bacterial genes by conjugation. F plasmid is an episome and may exist as a circular plasmid.
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What is an R plasmid?
Resistance factors - self mobile, can transfer between unreleated bacterial species. Encodes resistance to one or more antimicrobial drugs, heavy metals or toxins. Evolved with the widespread use of antibiotics.
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What are col plasmids?
Small plasmids, encode biological factors (e.g. colicin). Relaxed replication. Do not encode functions permitting transfer between individual bacteria - can be transferreed if F or R plasmids present in same cell, encoding functions for transfer.
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How were F plasmids discovered in E.coli?
Lederberg and Tatum - mixed 2 auxotrophic strains. Obseerved some prototrophic colonies when plated on minimal media. Conlonies resulted from genetic exchange.
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Which experiment showed that physicalcontact between strains was required for genetic exchange?
U Tube
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What is the mating system of E.coli termed?
Conjugation
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What is the process of bacterial conjugation?
A F+ cell contacts an F- cell - initial connection estabilished by a long tubular F pilus. F+ pilus contracts forming a bridge. Genetic material transferred through cytoplasmic bridge. F plasmid carries Tra (transfer) genes.
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What do the tra genes do?
They're used for contacting and mobilisation functons - encodes pilin proteins to build pilus.
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How are F plasmids transferred across two F genes?
F- pilus connect the two F cells. F+ pilus contracts forming a cytoplasmic bridge. Nick in the OriT of donor (F+) cell, circle replication occurs pa**ing the ** DNA from the 5' end to the F- cell. DNA synthesis in F- recipient restores second strand
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What is a Hfr strain?
High frequency strain
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What can form a high frequency transfer?
F plasmid can integrate into host chromosome to form an Hfr strain. This is done by recombination between insertion sequences on F plasmid and chromosome.
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Is F plasmid integration reversible?
Yes
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Transfer of bacterial chromoosmes first requres F factor to become...
Integrated into bacterial chromosomes
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What is the process of a high frequency transfer?
F plasmid integrated into chromosome via rolling circle replication. Becomes double stranded. Transferred chromosomal DNA recombines with recipient chromosome.
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What do Hfr strains conjugate with?
F- strains via unidirectional, ordered transfer of chromosomal genes from donor to recipient.
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Why does only a small part of the Hfr strain get transferred?
Because the bridge is very fragile and breaks - rare for more than a fraction of chomosome to be exchanged.
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What happens to transferred DNA of conjugation?
It is ether integrated into recipient chromosome or degraded
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How can conjugation be used to map bacterial genes?
Mix bacteria and then at various times after mating commences, withdraw samples, break mating cells apart and plate bacteria on selective media to determine which genes have been transferred from Hfr to F-.
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What are the differences between HfrH and F- in the experiment we did?
Hfr was prototropic resistant to sodium azide, infection with bacteriophage T1, sensitive to streptomycin, while F- was auxotrophic for threonine and leucine, sensitive to sodium azide and to infection by T1 and resistant to Streptomycin.
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How can we tell that Ecoli genetic map is circular?
Using a variety of Hfr strains that overlap.
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What does imprecise excition of F plasmid from bacterial chromosome generate F- plasmid carying bacterial genes
F plasmid integrated into chromosome. F factor loops out incorrectly, including a piece of chromosome. Single cross over generates F'lac+, an F factor including the lac+ region. This van transfer to recipient.
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Someone who recieves the F'lac+ will be...
Lac+/lac- partial diploid (merodiploid)
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What is transformation?
The uptake of Naked DNA as a method of gene transfer.
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How does transformation occur?
Isolation of DNA, with gragments of average size. Transformed DNA exchanged into (recombined) recipient genome.
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What can cotransformation frequency be used for?
To map bacterial gene orde
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Generalised transduction is mediated by...
Virulent bacteriophages e.g P1vir.
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What is the life cycle of a virulent bacteriophage
T4 phage uses its tail fibers to stick to specific receptor sites on the outer surface of an E.coli cell. The tail sheath contracts, thrusting a hollow core through the wall, injecting DNA into the cell. The cells DNA is hydrolyzed.
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What is the next stage of the life cycle of a bacteriophage?
The cell's metabolic machinery produces phage proeins and nucleotides from the cell's degraded DNA are used to make copies of the phage genome. The phage parts come together. The phage then directs production of lysozyme, and bursts.
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What happens when the cell bursts?
It releases 100 to 200 phage particles
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How does bacteriophage P1 mediate gene transfer of any pert of bacterial genome by generalised transduction?
Infection of donor bacterium with P1. Phage reproduction. Assembly of progeny wild-types and transducing phages. Some progeny phages package bacterial genes in heads. Release by lysis. Some phages are transducing phages, carrying donor bacterial DNA
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What happens to the transducing phages carrying donor bacterial DNA?
They infect recipient bacterium, releasing their DNA into the cell. Genetic exchange of donor a gene with recipient a gene by double crossover = stable transduced bacterium by recombination.
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What is cotransduction?
When genes are sufficientyl close, a single P1 can carry one or the other, or both. Recombination that incorporates both genetic markers is cotransduction
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Cotransduction frequency can be used to...
Map genes. Genes that are closer together are co-transduced more frequently than genes that are far apart. Frequency with which genes are cotransduced allows determination of gene order. Limit of cotransduction approx 100kbp.
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How is gene transfer by transductino done?
Specialised transduction mediated by temperate bacteriophages e.g. have both lytic and lysogenic life cycles.
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Linear molecules of ʎ DNA have sticky ends known as?
Cos (cohesive ends)
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What are the two possible fates by circularised ʎ DNA?
Lytic or lysogenic
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What is the lytic pathwy of circularised ʎ DNA?
It is replicated by rolling circle mechanism to produce a concatamer of linear DNA. Concatamer cut at cos sequence by ter enzmye to produce ʎ gnome sized molecule , packaged into phage. Cell later lyses
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What is the lysogenic pathway of circularised ʎ DNA?
Circular ʎ DNA integrates into bacterial genome at specific site - conservative site specific recombination (CSSR). It is now a prophage. Prophage replicated with host genome. Bacterial strains with prophage integrates are called lysogens. I
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In response to certain triggers, prophage can excise from host genome and...
Enter lytic pathway.
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Where does the bacteriophage lambda integrate into the E.coli chromosome?
At the attB site to form a lysogen
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Where does recombination occur?
Between the bacterial attB site and the attP site in the lambda DNA.
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Both attP and attB contain...
A spacer region, O, that is flanked y different integrase binding sites (B,B', and C,C').
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Site specific recombination occurs within..
Sequence O - the core sequence common to both att sites.
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How is the integrated prophage normally excised from chromosome by recombination between core O sequences of attL (BOC) and aatR (COB)?
Production of initial low frequency transducing (LFT) lysate when induction of lysogenic bacterium causes outlooping. Normal outlooping produces normal ʎ phages.
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What does rare abnormal outlooping produce?
Transducing ʎd gal+ phages. This is where some prophage DNA remains in bacterial chromosome, and excised bacteriophase lacks some prophage genome (defective) but carried bacterial DNA from region flanking integration site.
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What are the two possibilities when a mixed phage lysate infects E.coli?
If both integrate, an unstable transductant (double lysogen) results. Equal numbers of both are produced. Or Just a stable transuctant produed by recombination infection = un able to replicate as not all genes present.
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What are transposons?
They are transposable elements - pieces of DNA that can move around the genome and insert at target sites by transposition.
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Insertion of a transposon in a gene will do what?
Knock out it's function - they are a major source of mutation.
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Transposon insertion can affect...
Genes, gene regulation, chromatin structure, genome stability and evolution.
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What are the two ways in which transposons can move?
1.) Excision and integration 2.) Replication.
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Do transposons require sequence homology for insertions?
No
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What do transposon insertions result in?
They result in target site duplications - small sequences at the insertion site that become duplicated due to DNA repair mechanisms.
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What are the 3 main types of transposon?
DNA only cut and paste element, LTR element and Non LTR element
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What are DNA only cut and paste transposons?
They have terminal inverted repeats (TIR) that are recognized by the encoded transposase.
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What do autonomous transposons do and what do non autonomous transposons do?
Autonomous transposons encode the proteins needed to move the DNA. Non autonomous transposons rely on proteins made by an autonomous element.
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Where are DA only cut and paste transposons found?
Widespread in bacteria and eukaryotes. Within all organisms.
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DNA only transposons are the only type of transposon found in...
Bacteria
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DNA only transposons always contain an element bordered by..
Short inverted repeats that are recognised by element encoded transposase.
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Bacterial elements carry other genes such as..
Pathogenicity factors and genes for antibiotic resistance (this has continubted to spread of antibiotic resistance in bacteria). While Eukaryotic DNA only elements usually only contain the transposase.
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What are LTR elements?
Long terminal repeat elements are retroviruses. They encode several proteins including a reverse transcriptase.
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What are non LTR elements?
Can be autonomous or non autonomous and encode proteins with a range of activities.
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What are the autonomous nonLTR elements?
LINE (long interspersed elements) encode proteins that mediate thier own transposition (common in the human genome)
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What are the non autonomous nonLTR elements?
SINE - Short interspersed elements. Do not encode proteins for their movement and rely on those from LINEs.
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Bacterial DNA cut and paste elements that encode their own transposition functions are called...
Insertion sequences (IS elements)
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What is an insertion sequence?
It codes for enzyme needed for transposition, flanked by short inverted terminal repeats.
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Transposons have... and generate...
Inverted repeats and target repeats
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How do composite transposons work?
A pir of IS elements flank another gene, and the whole segment of DNA can act as a single trnsposable emenent. These often carry genes for drug resistance.
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What are the bacteria DNA only transposons which are non composite?
TrnA family
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What is the mechanism of transposition?
Most DNA only transposons move by cut and paste mechanism.
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What happens during the cut and paste mechanism?
The element excises completely and inserts into the target, using a small amount of replication to repair the join sites.
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What is Nick and paste movement and where is it found?
In bacteria only. In nick and paste, the transposon remains attached to the donor DNA and is joined to the target (forming a cointegrate which is eventually resolved into two molecules, each containing a transposon.
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What are transpososomes?
Elaborate protein DNA structures which contain the transposon DNA, the transposase and host proteins that may be required for transposition.
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What type of transposition occurs in transpososomes?
DNA cut and paste transposition.
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How does transposition occur at the molecular level?
DNA cut nd paste transposition occurs in transpososome. Terminal inverted repeat DNA recognised by transposase. Transposases oligomerize, bringing transposon ends togeth activating transposon cleavage from background DNA.
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What happens to the transposase/cleaved transposon complex?
It binds to target DNA and the 3' end attack target DNA and join at staggered positions. Single stranded DNAs are filled by host repair.
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How are Hfr stains generated?
By recombination between transposons on G plasmid and bacterial chromosome. Recombination between IS sequences on chromosome and plasmids can result in plasmids being integrated into host chromosome. Plasmid integration is reversible
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