Exchange of genes between two DNA molecules to form new combinations of genes on a chromosome contributes to a population’s genetic diversity (source of variation in evolution) Recombination is more likely than mutation to be beneficial Less likely destroy a gene's function May bring together combinations of genes

1. By
Assist.Prof
Dr. Berciyal Golda. P
VICAS
Genome Shiffling

2. Genetic Recombination
Exchange of genes between two DNA molecules to
form new combinations of genes on achromosome
contributes to apopulation’s genetic diversity (source
of variation in evolution)
Recombination is more likely than mutation to be
beneficial
Less likely destroy agene's function
May bring together combinations of genes

10. Double-strand breaks in DNA
initiate recombination
(part I)

11. Double-strand breaks in DNA
initiate recombination
(part II)

14. SITE SPECIFIC RECOMBINATION
(SSR)

15. 2. Site specific
recombination
Viruses and transposable elements often
integrate their genomes into the host
chromosome
Site specific recombination is used by both
eukaryotes and prokaryotes to regulate gene
expression and to increase the organisms
genetic range

16. Site specific
recombination
FIGURE 25–38 A site-specific
recombination reaction. (a) The reaction is
carried out within a tetramer of identical
subunits. Recombinase subunits bind to a
specific sequence, often called simply the
recombination site. 1 One strand in each
DNA is cleaved at particular points within
the sequence. The nucleophile is the OH
group of an active-site Tyr residue, and the
product is a covalent phosphotyrosine link
between protein and DNA. 2 The cleaved
strands join to new partners, producing
a Holliday intermediate. Steps 3 and 4
complete the reaction by a process similar
to the first two steps. The original sequence
of the recombination site is regenerated
after recombining the DNA flanking the
site. These steps occur within a complex of
multiple recombinase subunits that
sometimes includes other proteins.

17. WHY SSR???
• Transformation through Agrobacterium & direct
DNA transfer leads to complex integration of
GOI.
• Complex integration is classified into,
 Single copy – multi locus integration
 Multi copy – single locus integration
• Complex integration leads to gene silencing.

18. • Ideal integration is single copy – single locus and
integration of GOI without intervening with
functional gene.
• The best way out of this issue is promoting the
development of system/technique that facilitates
site specific recombination.

19. • Two systems have been developed for
facilitating site specific recombination,
namely,
Cre/lox system
FLP/FRT system

20. Cre/lox system
• Derived from P1 bacteriophage.
• Cre recombinase catalyses the recombination
between two loxP(site for recombination) sites.
• loxP site consists of an 8-bp core sequence, where
recombination takes place, and two flanking 13-bp
inverted repeats.(In total 34-bp)

21. loxP site

22. The Jackson Laboratory

23. FIGURE 25–39 Effects of site-specific recombination. The outcome of site-specific
recombination depends on the location and orientation of the recombination sites (red
and green) in a double-stranded DNA molecule. Orientation here (shown by arrowheads)
refers to the order of nucleotides in the recombination site, not the 5n3 direction.
(a) Recombination sites with opposite orientation in the same DNA molecule. The
result is an inversion. (b) Recombination sites with the same orientation, either on one
DNAmolecule, producing a deletion, or on two DNAmolecules, producing an insertion.

24. FLP/FRT system
• Derived from 2 micrometer yeast plasmid
• FLP recombinase enhances recombination of
sequences between two short Flippase
Recognition Target (FRT).
• FRT consists of an 8-bp spacer and two flanking
13-bp inverted repeats, where recombination
takes place (In total 34-bp).

25. FRT site

26. DNA Transposition
recombination that allows the movement of transposable
elements, or transposons. These segments of DNA, found in
virtually all cells, move, or “jump,” from one place on a
chromosome (the donor site) to another on the same or a
different chromosome (the target site). DNA sequence
homology is not usually required for this movement, called
transposition; the new location is determined more or less
randomly. Insertion of a transposon in an essential gene could
kill the cell, so transposition is tightly regulated and usually
very infrequent. Transposons are perhaps the simplest of
molecular parasites, adapted to replicate passively within the
chromosomes of host cells. In some cases they carry genes
that are useful to the host cell, and thus exist in a kind of
symbiosis with the host

27. Bacteria have two classes of transposons.
1. simple transposons
Insertion sequences contain only the sequences required for transposition and the
genes for proteins (transposases) that promote the process.
2.Complex transposons contain one or more genes in addition to those needed for
transposition. These extra genes might, for example, confer resistance to antibiotics and
thus enhance the survival chances of the host cell. The spread of antibiotic-resistance
elements among disease-causing bacterial populations that is rendering some antibiotics
ineffectual is mediated in part by transposition. Bacterial transposons vary in structure, but
most have short repeated sequences at each end that serve as binding sites for the
transposase. When transposition occurs, a short sequence at the target site (5 to 10
bp) is duplicated to form an additional short repeated sequence that flanks each end of
the inserted transposon (Fig. 25–42). These duplicated segments result from the cutting
mechanism used to insert a transposon into the DNA at a new location.
Classes of Transposons

28. FIGURE 25–42 Duplication
of the DNA sequence at a
target site
when a transposon is
inserted. The duplicated
sequences are shown
in red. These sequences are
generally only a few base
pairs long, so their size
(compared with that of a
typical transposon) is
greatly exaggerated in this
drawing.

29. There are two general pathways for transposition in bacteria.
In direct or simple transposition (Fig. 25–43, left), cuts on each side of the
transposon excise it, and the transposon moves to a new location. This leaves a
double-strand break in the donor DNA that must be repaired. At the target site, a
staggered cut is made (as in Fig. 25–42), the transposon is inserted into the break,
and DNAreplication fills in the gaps to duplicate the target site sequence.
In replicative transposition (Fig. 25–43, right), the entire transposon is
replicated, leaving a copy behind at the donor location. A cointegrate is an
intermediate in this process, consisting of the donor region covalently linked to
DNAat the target site.
Two complete copies of the transposon are present in the cointegrate, both having
the same relative orientation in the DNA. In some well-characterized transposons,
the cointegrate intermediate is converted to products by site-specific
recombination, in which specialized recombinases promote the required deletion
reaction.

30. FIGURE 25–43 Two general pathways for
transposition: direct (simple) and replicative. 1
The DNA is first cleaved on each side of the
transposon, at the sites indicated by arrows. 2 The
liberated 3- hydroxyl groups at the ends of the
transposon act as nucleophiles in a direct attack on
phosphodiester bonds in the target DNA. The target
phosphodiester bonds are staggered (not directly
across from each other) in the two DNA strands. 3
The transposon is now linked to the target DNA. In
direct transposition, replication fills in gaps at each
end. In replicative transposition, the entire
transposon is replicated to create a cointegrate
intermediate. 4 The cointegrate is often resolved
later, with the aid of a separate site-specific
recombination system.
The cleaved host DNA left behind after direct
transposition is either repaired by DNA end-joining
or degraded (not shown). The latter outcome can be
lethal to an organism.

31. Genetic Transfer
Vertical genetransfer
From parents to offspring
Horizontal genetransfer
From one microbe to another
Between different strains and species of
bacteria and viruses
Leads to recombination

32. Horizontal gene transfer
Part of total DNA from Donor cell integrated into
Recipient cell.
Remaining amount of DNA from donor cell
degraded.
Recipient cell with DNA from donor is called
Recombinant.
1% of population might undergo recombination

33. Recombination in prokaryotes occurs
through three mechanisms
1. Transformation
2. Transduction
3. Conjugation
Horizontal gene transfer -
Mechanisms

34. Transformation
Transfer of naked DNA from donor to recipient
cell
Discovered by Frederick Griffith in 1928 in
Streptococcus pneumoniae
Showed that DNA is the genetic material
Can be transferred between a donor and a recipient
cell
Griffith’s expt: Avirulent S. pneumonia
became virulent when exposed to heat killed
virulent cell

35. Transformation experiment by
Griffith

36. Bacterial transformation without mice
Broth containing non-encapsulated living
bacteria and dead encapsulated bacteria
incubated
After incubation, encapsulated living virulent
bacteria were found
Non-encapsulated bacteria received genes from
dead encapsulated for forming a capsule
The material responsible for transmission of
this character was not known

37. Experiment: DNA is the genetic material
In 1944, Oswald T Avery, Colin M Macleod,
Maclyn Mccarty proved that DNA is the genetic
material

38. Competent cells & competence
Competence: ability of a recipient bacterium to
take up DNA from the environment
Competent cells: cells which can be transformed
E.coli cannot undergo transformation naturally
It is made competent by artificial transformation
procedures (Calcium chloride or Electroporation)

39. Mechanism of transformation
After death, cell lysis leads to release of DNA from
bacteria
Other bacteria take up DNA and integrate into their
chromosomes by recombination
Recipient cell with this combination of genes will now
become a hybrid or recombinant
Works best between closely related species
Transformation in nature: Bacillus,Haemophilus,
Streptococcus,Staphylococcus,Neisseria etc.

40. Mechanism of transformation
Step1:
The DNA binding receptor on a competent
bacterium binds double stranded DNA
As the DNA enters the cell, one strand is
degraded, & the other strand is coated with single-
strand DNA-binding protein.
Step2:
The single strand of donor DNA is integrated into
the chromosome of the recipient cell producing a
recombinant DNA

41. Mechanism of transformation

42. Conjugation
Transfer of genes between cells that are in
physical contact with another
First demonstration of recombination in bacteria:
Jhosua Lederberg & Edward Tatum in 1946
Found that, genetic traits could be transferred
among two different strains of E. coli, if they are
in physical contact

43. F+ and F- FACTORS
William Hayes, Francois Jocob and Eli H Wolman
(1950)
Conjugating bacteria are of two mating types:-
Male types which donates their DNA, these are
called F+ cells
Female types which are recipient of DNA
donated by F+ cells and are called F- cells
These F+ and F- are called fertility factor or F-
factor or sex factor

44. Process of Conjugation
The F Pili of the F+ donor cell make contact
with the F- recipient cell & pull the cell together.
Rolling circle replication transfer one strand of
the F factor into the recipient cell.
Transfer of F factor is completed, yielding two
F+ factor bacteria.

45. Process of Conjugation

46. Process of Conjugation
In donor F+ cells, F factor may integrate into the
host chromosome becoming Hfr (High
Frequency of Recombination).
Thus F+ cells become Hfr cells
Conjugation between Hfr and F- cells results in
replication of the chromosome with F factor.
Asingle parental strand is transferred from Hfr
cell to the F- cells.

47. Process of Conjugation

48. Process of Conjugation
Complete transfer of the chromosome does not take
place
Only a small piece of F factor leads the chromosomal
genes into F- cells
Small strand containing chromosomal genes
recombines with the DNA of F- cells
Thus F- cells receive only a part of chromosomal
genes and hence do not get converted to F+ cells

49. Transduction
Transduction occurs when a phage (virus) carries
bacterial genes from one host cell to another
Discovered by Norton Zinder and Joshua
Lederberg in 1952
Bacteriophage Twotypes:
1. Bacteriophage T4
2. Bacteriophage λ
Lifecycle
1. Lytic cycle
2. Lysogenic cycle

50. Lytic & Lysogenic Cycle

51. Lytic & Lysogenic Cycle
Bacteriophage attaches to donor bacteria
Inject their nucleic acid (DNA) into bacterium
DNA replicates rapidly, and also directs the synthesis
of new phage protein
Then, the new DNA combines with new proteins, to
make whole phage particles
These are then released by destruction of cell wall and
lysis of the cell

52. Process of Transduction
These phages may composed of DNA of the host
This phage attacks the another host and infect it
Recipient DNA integrates with this DNA
Results in the transfer of DNA
Recipient cell is now called transducedcell

53. Types of Transduction
Mainly there are two types
Generalised or Non-specialised Transduction
Restricted or Specialized Transduction

54. Generalized Transduction
All fragments of bacterial DNA have a
chance to enter a transducing phage

55. Specialized Transduction
Certain phages can transfer only a few restricted
genes of the bacterial chromosomes
Only those bacterial genes adjacent to prophage
in bacterial chromosomes
Mediates the exchange of only limited numbers
of specific genes
Mediated by Bacteriophage λ

56. Specialized Transduction
λ phage can only
incorporate into a specific
site (attλ)
gal gene is on one side of
attλ and bio gene (biotin
synthesis) is on the other
side
Wrong cross over of λ
phageat the end of the
lysogenic phase
Piece of the E. coli
chromosome incorporated
into λ phage chromosome
gal gene or bio gene can be
transferred