BTY 227 Molecular and Environmental Biotechnology Lecture 3 RECOMBINATION AND GENE TRANSFER - PowerPoint PPT Presentation

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BTY 227 Molecular and Environmental Biotechnology Lecture 3 RECOMBINATION AND GENE TRANSFER

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Transformation: involves donor DNA free in environment ... Contact via a protein tube called a F or sex pilus. 3 types: Basic. HFr. F' (F prime) ... – PowerPoint PPT presentation

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Title: BTY 227 Molecular and Environmental Biotechnology Lecture 3 RECOMBINATION AND GENE TRANSFER


1
BTY 227Molecular and Environmental
BiotechnologyLecture 3 RECOMBINATION AND GENE
TRANSFER

2
  • This lecture
  • Introduces bacterial recombination
  • Details the events that precede bacterial
    recombination
  • Conjugation
  • Transformation
  • Transduction

3
Definition of Recombination
  • The process by which parts or all of the DNA
    molecules from 2 separate sources are exchanged
    or brought together into a single unit
  • 3 types homologous (general)
  • site specific and
  • replicative recombination

4
Why learn about Recombination?
  • Recombination is an important evolutionary
    process
  • Through this process new combinations of genes
    can arise even in the absence of mutation
  • Mutation generally brings a small genetic change
    while recombination can involve much larger
    changes
  • Can result in adaptations to new environments

5
Homologous Recombination
  • Prior to this process a homologous DNA fragment
    must be transferred to a recipient cell
  • genetic exchange between the homologous DNA from
    two different sources occurs
  • Look at one recombination mechanism - reciprocal
    homologous recombination

6
Reciprocal HR (1)
  • A nuclease nicks the donor strand
  • Single stranded binding protein binds
  • RecA protein binds and forms a complex that
    allows annealing with complementary sequence on
    recipient DNA at the same time displacing the
    resident strand
  • Pairing can occur over 100s or 1000s of bases

7
Reciprocal HR (2)
  • Following pairing, exchange of homologous DNA
    molecules can occur
  • This leads to formation of recombination
    intermediates containing extensive heteroduplex
    regions
  • These are resolved by nucleases and DNA ligase
    to form two recombinant molecues

8
Detection of Recombination
  • To detect recombinants, they must be
    phenotypically different from the parent cells
  • E.g. trp- parent cell which undergoes
    recombination with DNA from a trp donor cell
    detect recombinants on selective medium that
    lacks tryptophan

9
Transfer of DNA between Cells
  • In prokaryotes DNA transferred to recipient cell
    by 1 of 3 processes
  • Transformation involves donor DNA free in
    environment
  • Transduction donor DNA transfer mediated by a
    virus
  • Conjugation involves cell to cell contact
    between donor and recipient i.e. acquisition
    directly from another bacterium. 3 types Basic,
    HFr and F

10
Transformation
  • Cells pick up DNA from the environment
  • In order to become transformed cells must be
    COMPETENT i.e. they must be able to transport the
    exogenous DNA into the cell
  • Exogenous DNA binds to the cell surface
    receptors and are transported across the membrane
  • 1 strand digested away by exonuclease
  • non-reciprocal HR follows (mediated by recA
    protein)

11
Conjugation
  • A mating process involving bacteria
  • Requires physical contact between cells
  • Contact via a protein tube called a F or sex
    pilus
  • 3 types
  • Basic
  • HFr
  • F (F prime)

12
Basic conjugation
  • involves 2 strains of bacteria F and F- -
    difference betw them is presence of F(ertility)
    factor
  • F factor is an episome (conjugative plasmid) that
    contains 19 genes which confer ability to
    conjugate on donor cell
  • Genetic material transferred is one strand of
    theF factor itself
  • Final result 2 F cells no recombination

13
Conjugation involving Hfr Bacteria
  • Occasionally the F factor integrates into a
    random position in host chromosome
  • Host cell then called Hfr (not F)
  • Hfr cells can still initiate conjugation but
    outcome completely different
  • Hfr cell tries to transfer entire bacterial
    chromosome to F- cell
  • Transfer of chromosome almost never complete and
    F- cell remains F-
  • New DNA in recipient can undergo recombination
  • Result Hfr and F- cell

14
F Conjugation (1)
  • Integrated F factors can occasionally excise from
    host chromosome
  • If excision is accurate F cells result, but
    sloppy excision leads to F cells where some of
    the chromosomal DNA excises with the F factor
  • Fcell has full complement of chromosomal genes
    but some are now on the plasmid
  • F cell can conjugate with F- cell (F factor
    present)

15
F Conjugation (2)
  • Both donor and recipient end up with copy of
    episome
  • F- now F but it is diploid for the segment of
    chromosomal DNA on the episome now F
    merozygote
  • These regions of DNA undergo recombination at
    high frequency
  • Result F and F merozygote

16
Summary of 3 Methods of Recombination
  • F mating with F- produces 2 F
  • Hfr mating with F- produces Hfr and F-
  • F mating with F- producesF and F merozygote
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