Insertion Sequences, Transposon, And Integrons 2 Flashcards
Creating mutants using transposons; often with suicide vectors
Transposon mutagenesis
Properties of transposons for mutagenesis
Transpose at a fairly high frequency
Not be very selective in its target sequence
Should carry an easily selectible gene (ex. Antimicrobial resistance)
Have a broad host range for wide range of applicability
Limitations of in vivo transposon mutagenesis
- Transposon should be introduced to suicide vector or else errors will be encountered if the vector replicates
- Possibility of multiple transposition
events if it lacks target specificity - Too many mutations encountered in the chromosome
- Plasmid or smaller DNA sequence is to be mutated
Target DNA can either be _____ (or plasmids that replicate by themselves) or random linear pieces of the host chromosomal DNA that can be introduced by transformation
Replicons
Tn5 and Himar1 originally from the horn fly ______; And has a _____ resistance gene
Haematobia irritans; Kanamycin
Transposons with the transposase already attached are called
Transpososomes
True or False: Transposon mutagenesis can be used for non-essential genes to avoid killing the host
True
Only between specific sites of DNA
Site specific recombinases
are site-specific recombinases which end up with the two sequences integrating with each other
Integrases
Enumerate the steps for site specific recombinases
1.Excise the DNA in the two
recognition sites:
a. If in same DNA, resolvases and
invertases
b. Integrases if from different DNA
2. Join the cut ends while making sure the recognition sites meet each other.
3.Hold and orient the DNA to meet recognition sites.
integrate the phage DNA to
host DNA
Phage integrase
Example of Phage Integrase
Int enzyme of lambda phage
Recognized sites for integration of lambda phage
- attB site of host DNA
- attP site of phage DNA
Enzymes involved in lambda phage
Integrase (Int)
Excisase (Xis)
similar regions in transposons that contain antibiotic resistance genes
Integrons
Integrons that are found in chromosome
Superintegrons
Parts of Integrons
- Integrase gene (intl1) that
facilitates recombination - Attachment site (attI)
recognized by integrons for
recombination and often next
to intl1 - Promoter (pc) for transcription
of the genes within the integron
and next to attI
are short DNA segments inserted into integrons or small circular DNA
Cassettes
Large segments of DNA often composed of several genes related to pathogenicity
Genetic pathogenicity islands (PAIs)
Genes responsible for toxic shock syndrome in Staphylococcus aureus
PAI SaPI1
True or False: PAIs cannot be transferred via phages such as phage 80a
False
Resolves cointegrates of
transposition by recognizing the
res sequence
Resolvases
True or False: Resolvases are applicable to plasmids in dimer formation
True
promote site-specific recombination but the two DNA sites are in reverse orientation in relation to each other (invertible sequences); Useful for evading immune responses and is reversible
DNA invertases
Salmonella H1 to H2 flagellin often invert using the ______ through recombination between the sites hixL and hixR.
Hin invertase
Either a tyrosine (Y) or serine (S) is in the active center of the
recombinases
Y and S recombinases
____ recombinase that recognizes loxP site for Y recombinase
Cre
Y and S recombinases form what junction? 3’-ends join the 5’-ends
Holliday junction
