13/ bacterial genes 3

Question 1

name 3 ways dna from a donor cell can be introduced into recipient cell

Answer 1

• transformation - uptake of naked dna
• transduction - phage mediated
• conjunction - plasmid mediated

Question 2

mechanism of transformation

Answer 2

• naked dna binds via protein
• one strand enters using dna translocase, 2nd strand degraded
• internalised strand bound by RecA protein
• RecA has more than 1 dna binding site, so can hold ssDNA and dsDNA together
• RecA-ssDNA complex stretches the dsDNA to increase complementarity recognition
• branch migration and homologous recombination follows
• in lab need to do stuff eg electrocute to allow dna to enter

Question 3

mechanism of generalised transduction

Answer 3

• normal bacteriophage contains phage genes, transducing particles also contain host genes
• phage accidently puts bacterial genes into its genome and carries it around
• because dna is bacterial if successful, bacteria wont destroy it

Question 4

mechanism of conjugation - fertility F plasmid background in e coli

Answer 4

• only in donor cells
• includes insertion sequence which can recombine w regions on chromosome
• origin of transfer during replication
• genes needed for conjugative transfer, inc sex pilus

Question 5

what is a pilus

Answer 5

• involved in cell pairing for mating in bacteria
• long projection that grabs onto another bacteria

Question 6

mechanism of conjugation - transfer of F fertility plasmid

Answer 6

• cells get closer together til they touch using pilus
• keep 1 copy of plasmid and transfer other by
• nick ds plasmid - one strand starts moving towards the F- bacteria (w/o F plasmid)
• recipient bacteria ds the ss F
• now there are 2 F+ bacteria

Question 7

how can chromosomal genes be transferred

Answer 7

• integrated F plasmid
• isolate High frequency of recombination Hfr strains
• insertion sequence identical to sequences in bacterial chromosome where F plasmid is, if they come together recomb will happen
• plasmid in Hfr no longer controls its own replication but the tra function operates and cell forms pili
• origin of transfer oriT nicked and dna starts passing to neighbouring cell. whole of F plasmid must be transferred to make a … (pili genes too)
• bacteria w free F plasmid is F+, when this gets integrated into chromosome becomes Hfr
• Hfr strains can transfer as efficiently as F+

Question 8

how are transformation, transduction and conjugation used in labs

Answer 8

• transformation: widely used to get plasmids into bacteria. some bac naturally competent, others need to be shocked into accepting dna
• transduction: 2nd most common. move smaller fragments on dna
• conjugation used to be widely used, not so much now