Lab 3

Question 1

Transposons

Answer 1

• a DNA sequence that can change its position within a genome
• sometimes creates or reverses mutations and alters the cell’s genetic identity and genome size

Question 2

Transposition

Answer 2

• often results in duplication of the same genetic material
• can be classified as either “autonomous” or “non-autonomous”
  
  • Autonomous transposons can move by themselves
  • non-autonomous transposons require the presence of another TE to move.
    
    • often because the dependent transposons lack transposase or reverse transcriptase

Question 3

Simple transposons, insertion sequences (IS elements)

Answer 3

• small relative to other transposable elements
• Autonomous
• code only for protein(s) required for transposition
  
  • proteins coded
    
    • transposase which catalyses the enzymatic reaction allowing the IS to move
    • regulatory protein which either stimulates or inhibits the transposition activity
  • different from other transposons, which also carry accessory genes such as antibiotic resistance genes
• The coding region in an insertion sequence is usually flanked by inverted repeats.
  
  • consisting of inverted terminal repeats (of specific sequence)
  • usually between 15 and 25 bp
  • not identical but closely related
• require a transposase for transposition
• are directional
  
  • from left to right, or right to left
  • in order to code for the genes in the middle
  • denoted by red arrow in image
• may also occur as parts of composite transposons
  
  • in a composite transposon, two insertion sequences flank one or more accessory genes, such as an antibiotic resistance gene

Question 4

Composite transposons

Answer 4

• denoted as “Tn” with a number (eg. Tn5, Tn9, Tn10);
  
  • Tn5 will be used in the lab
• has protein coding DNA segments
  
  • often carry one or more genes conferring antibiotic resistance
  • antibiotic resistance can be used as a marker for the transposases
• flanked by two separate IS elements
  
  • IS element ends are referred to as arms or IS modules
  • can be direct or inverted repeats
  • can be identical or closely related
    
    • if identical, either module, can sponsor the movement
    • If different, the protein encoding the transposase may differ, and only one module may function correctly;
  • Instead of each IS element moving separately, the entire length of DNA spanning from one IS element to the other is transposed as one complete unit
• transposition
  
  • transposase will move any segment of DNA surrounded by a pair of the inverted repeats that it recognizes
  • several possibilities.
    
    • each of the insertion sequences may move independently
    • the whole structure between the two outermost inverted repeats may move as a unit, that is, as a composite transposon

Question 5

Transposon insertion sites

Answer 5

• insert into specific sites based on sequence
• most insertion sites sequences can be flexible,
• the length of the recognized insertion site sequence is usually 5 to 9 bp
• insertion sequence specificity leads to hot or cold spots in random insertions
• the insertion site results as a direct repeats
  
  1. insertion site is found due to specificity of sequence to the transposon/transposase
  2. the host DNA is cut at the insertion site creating “sticky ends” with overhangs
  3. the transposon add complementary DNA sequence to overhangs
  4. transposon leaves and ligases the DNA back together
  5. Duplicate sequence/repeats are generated as a result

Question 6

Chemical mutagenesis

advantages / disadvantagesmajor advantages: – just about every organism is susceptible – Less likely to lead to hot spots (at least at the level of Tn mutagenesis. Why?) • Major disadvantages: – Difficult to determine the location of the mutation, and thus identify the mutated gene – Difficult to control the number of mutations per cell

Answer 6

• advantages
  
  • just about every organism is susceptible
  • Less likely to lead to hot spots as compared to transposons
• disadvantages
  
  • Difficult to determine the location of the mutation / identity of gene
  • Difficult to control the number of mutations per cell
  • multiple mutations make it difficult to identify which mutation is associated with the mutant phenotype of interest
    
    • this is why you want to limit the number of mutations

Question 7

Chemical mutagenesis

Identification of a mutated gene

Answer 7

• The standard method
  
  • complement the mutation by mobilization of a wt genomic library into the mutant
  • screen for restoration of the phenotype
  • strain must be genetically tractable
    
    • has to be amenable to accepting plasmids
  • labor intensive approach
• sequence the genome to find mutated nucleotide
  
  • requires high coverage and computational analysis
• biochemical characterization of the mutant
  
  • look for differences in protein profiles between mutant and wt
  • not mainstream
  • getting easier with high throughput systems and genomic/proteomics
    
    • cost associated with analysis

Question 8

Transposon Tn5

Answer 8

• One of the first transposons to be identified
• naturally occurring composite transposon, mainly found in enterics
• widely used because it is capable of inserting throughout most locations in the bacterial genome
• Resistant to the antibiotic Kanamycin and a few other antibiotics
• Encode two different proteins associated with transposition
  
  • related in sequence
  • Transposase
  • Transposition inhibitor
    
    • prevents movement of the transposon once it’s inserted
    • prevents other copies of Tn5 from inserting into the genome
    • does this by competitive binding to transposase binding sites
    • density dependent
      
      • repression of movement is relaxed once inhibitor is diluted
    • for research purposes
      
      • ensures there is one transposon per genome
      • limits number of mutations
      • stabilizes insertion, ensuring transposon is restricted in its movement

Question 9

miniTn5lacZ1Cat

Answer 9

• molecularly constructed composite transpositional element
• a derivative of Tn5
• not a true transposon
• It does NOT contain a transposase
  
  • why it’s not considered a transposon
  • cannot sponsor movement on its own
  • once it inserts, it stays there permenantly
• has IS element sequences at the end
  
  • inverted repeats
  • not an entire IS element
• has resistance to chloramphenicol
  
  • selective marker 1 for transpositional element
  • if transpositional element is inserted into strain, this follows
• has a promoterless lacZ gene
  
  • selective marker 2
  • located at one end of the element
  • used to allow transcriptional fusions to occur upon insertion
• contained in pUT plasmid

Question 10

pUT plasmid

Answer 10

• system used for delivery to recipient strain targeted for mutagenesis
• has the following
  
  • transposase
    
    • moves miniTn5lacZ1Cat from plasmid to genome
  • Ampicillin (Ap) resistance
    
    • functions as a selective marker for the plasmid
  • R6K origin of replication
    
    • requires π (pi) protein for replication
    • originated from λ phage
  • oriT (ori of transfer)
    
    • allows the plasmid to transfer from the donor to the target strain via conjugal mating
• remember that miniTn5lacZ1Cat has resistance to chloramphenicol
  
  • if plasmid has resistance to miniTn5lacZ1Cat then it still has the miniTn5lacZ1Cat within it
  • if plasmid is no longer resistant to chloramphenicol, then miniTn5lacZ1Cat has transpositioned (hopefully to the target strain)
• once plasmid has been transferred to the recipient strain
  
  • the plasmid’s transposase moves miniTn5lacZ1Cat from plasmid to genome
  • plasmid cannot replicate, because the recipient strain is missing λ pir gene that encodes the π protein for plasmid replication
  • plasmid eventually degrades

Question 11

E. coli strain S17-1λpir

Answer 11

• used to house pUT plasmid
• strain serves as the donor
• used to replicate and deliver pUT plasmid
• has the λ pir gene that encodes the π protein for plasmid replication
  
  • initiator protein
  • required for R6K origin of replication in pUT plasmid
  • recipient strains typically do not have the pir gene
    
    • so if pUT plasmid is inserted, it would not replicate in that strain and eventually go away (“suicide nature of plasmid”)
• supplies MOB/MPF functions encoded by genes in the chromosome
  
  • mobilization and mating pair formation genes
  • necessary to mobilize the pUT plasmid into the recipient strain through a matin channel/conjugate mating
  • MOB prepares plasmid for transfer
  • MPF build the channel for transfer
• recipient strain
  
  • plasmid replication is not desired in recipient
  • this ensures selection of chloramphenicol is because miniTn5lacZ1Cat transpositioned into the recipient’s genome and not because it was found in the unwanted replicated plasmids