This study evaluated the effect of expressing multiple heterologous recombinases on increasing homologous recombination in tobacco plants. The recombinases RecA, RecG, RuvC, Rad51, Rad52 and DMC1 were expressed individually and in combinations in tobacco plants containing a recombination substrate. Expression of DMC1 alone produced the greatest stimulation of homologous recombination, increasing recombination frequency up to 1000-fold. Expression of other recombinases also increased recombination 2 to 380-fold. Increasing homologous recombination could improve the efficiency of gene targeting for plant biotechnology applications using CRISPR/Cas.

1. RECOMBINATION MODELS &
CYTOLOGICAL BASIS OF CROSSING OVER
TERM PAPER PRESENTATION
GP-502
SUBMITTED TO:
Dr. D. Shivani
PROFESSOR
DEPT. OF GENETICS AND PLANT
BREEDING
SUBMITTED BY:
ANIL KUMAR
RAM/2020-68
GPBR

2. CONTENTS
Introduction
Types of recombination
Homologous or generalized recombination
Site-specific recombination
Transposition
Models for homologous
recombination
Models involving single strand breaks: Holliday
model
Models involving double strand breaks
Cytological basis of crossing over
Experiment of stern in drosophila
Studies Creighton and McClintock in Maize

3. INTRODUCTION
Recombination may be described as
production of new combination of linked
genes.
It occurs between precisely homologous
sequences in such a manner that not a
single base pair is added to or lost from
recombinant chromosomes.
Recombination involves physical exchange
of material between duplex DNAs.

4. THREE DIFFERENT TYPES OF
RECOMBINATION
1. Homologous or generalized
recombination
2. Site-specific recombination
3. Transposition

5. HOMOLOGOUS OR GENERALIZED
RECOMBINATION
Homologous recombination involves the
exchange of precisely corresponding
sequences between homologous DNA
duplexes.
In eukaryotes, it occurs during the four
strand stage of meiosis, involves only two
of the four chromatids of a bivalent.
It may occur at a lower rate in

6. SITE-SPECIFIC RECOMBINATION
Site-specific recombination occurs between
specific pairs of DNA sequences since the
enzyme involved can act only on a particular
pairs of target sequences.
This types of recombination has been
characterized in prokaryotes, e.g.,
integration of phage genome into bacterial
chromosome.

7. TRANSPOSITION
In transposition, breakage and reunion of
DNA strand allows one DNA sequence to be
inserted into another without reliance on
sequence homology.
The movable DNA segments called
transposons.

9. MODELS FOR HOMOLOGOUS
RECOMBINATION
Models involving single strand
breaks:
Holliday model
Meselson & Raddling Model
Models involving double strand
breaks:
Szostak-Orr-Weaver-Rothestein-Stahl

10. HOLLIDAY MODEL
Proposed by Robin Holliday in 1964.
According to this model, 1st an endonuclease
produce single strand nicks at identical points
in the two homologous parental DNA molecule
in the strands having same polarity.
Segment of single strands on one side of each
nick are then displaced from their
complementary strands up to some distance.
Displaced single strand then crossover and
pair with intact complementary strand of
homologous chromosomes producing a joint
molecule.

11. CONT…
Each duplex in a joint molecule has a region in
which it has one strand from each of the two
parental DNA duplexes, this region is called
hybrid DNA or heteroduplex DNA.
The two single strand nicks remaining in the two
duplexes are then joined by DNA ligase.
The joint molecule undergoes reorientation to
form an x-shaped structure called chi forms.
One end of chi form now rotates by 180⁰ to
form Holliday structure.
Holliday structure is resolved by enzyme
catalysed breakage and re-joining of
complementary DNA strands to produce two

13. CONT…
An endonuclease now induce single strand
nicks (in those strands that were not nicked
brfore) in the holliday junction.
As a result two splice recombinant DNA
duplexes will be generated.
The recombinant duplexes will have one nick
each, which is then sealed by DNA ligase.

14. MODELS INVOLVING DOUBLE STRAND
BREAKS
In 1983, J. Szostak and colleagues put forth a
different model, initiated by double-strand
break in one of the double helices. The DNA
duplex in which the break is induced is called
recipient duplex.
The break is induced by endonuclease followed
by 5’→3‘ exonuclease activity to widen the gaps
formed in the double helix and create 3 single-
stranded tails on both sides of the break.
One of the single stranded 3‘-end now invades
the homologous region of other DNA duplex,
called donor duplex.

15. Homologous strand in the donor duplex is
replaced by invading strand.
The displaced strand of donor duplex is now
pair with other single stranded 3‘- end of
recipient duplex.
Repair synthesis of previously digested DNA
using 3‘-end as a primer and strand of donor
duplex as template fills the gap.
Nicks remaining in two duplexes are sealed by
DNA ligase.
CONT…

17. Branch migration
converts the two DNA
duplexes into a joint
DNA molecule having
two recombinant
joints.
The two joints are
resolved in opposite
ways to produce a
recombination event.
CONT…

18. CYTOLOGICAL BASIS OF
CROSSING OVER
Experimental evidence of crossing over
was provided in 1931 independently by:-
Curt Stern in Drosophila &
Creighton and McClintock in Maize

19. EXPERIMENT OF STERN IN
DROSOPHILA
In his experiment, Stern used a drosophila female
in which one X chromosome was shorter than
normal.
This chromosome had a recessive gene car
(carnation eye colour) and dominant gene B (Bar
eye shape).
The other X chromosome of this female was of
normal length, but a segment of Y chromosome
was translocated into short arm.
This chromosome had the dominant gene car+

20. Stern test crossed this female to a car B+
(carnation normal) male.
As expected the following four types of
flies were recovered in test cross progeny:
Red, normal (car+ B+)---Parental type
Red, bar (car+ B)---Recombinant type
Carnation, normal (car B+)---Recombinant
type
Carnation, bar (car B)---Parental type
(Allele contributed by the test cross parent are not
shown)
CONT…

21. If crossing over involves exchange of sister
chromatid between homologous chromosome
then one X chromosome of recombinant
individual would be the product of such
exchange.
Therefore Carnation, normal (car B+) flies are
expected to have a normal X chromosome
without the attached Y chromosome.
Red, bar (car+ B) will have one short X
chromosome with attached Y segment.
CONT…

22. Stern observed very close correspondence
between expectation and results actually
obtained.
He concluded that:
During meiosis, there is exchange of precisely
homologous chromatin segment between
homologous chromosome.
Crossing over is responsible for
recombination between linked genes.
CONT…

24. STUDIES CREIGHTON AND
MCCLINTOCK IN MAIZE
A similar conclusion was reached by Creighton
and McClintock from their study in Maize.
They used a maize plant in which one
chromosome 9 was normal and had recessive
gene c (colourless aleurone) and dominant gene
Wx (nonwaxy endosperm).
The chromosome 9 of this plant had a knob and
was involved in unequal reciprocal translocation
with chromosome 8.
This chromosome had the dominant gene C
(coloured aleurone) and the recessive gene wx

25. This plant was testcrossed with the double
recessive strain (c wx/c wx) having normal
chromosome.
The phenotype as well as morphology of
chromosome 9 of test cross progeny were
recorded.
The data were in prefect agreement with
the expectation as was the case in
Drosophila experiment.
CONT…

26. CASE STUDY

27. Name of Journal: BMC Plant Biology
NAAS Score: 9.67
Received: 18 November 2019 Accepted: 8 July 2020
Published online: 16 July 2020

28. BACKGROUND
Current excitement about the opportunities for
gene editing in plants have been prompted by
advances in CRISPR/Cas and TALEN technologies.
CRISPR/Cas is widely used to knock-out or
modify genes by inducing targeted double-strand
breaks (DSBs) which are repaired predominantly
by error-prone non-homologous end-joining or
microhomology-mediated end joining.
Gene replacement (or gene targeting) by
homology directed repair occurs at extremely low
frequency in higher plants making screening for
useful events unfeasible.

29. • Homology directed repair might be increased by
inhibiting non-homologous end-joining and/or
stimulating homologous recombination (HR).
• Here we pave the way to increasing gene
replacement efficiency by evaluating the effect of
expression of multiple heterologous recombinases
on intrachromosomal homologous recombination
(ICR) in Nicotiana tabacum plants.
CONT…

30. METHODS
• Bacterial and human recombinases cloning:- The
coding sequences of bacterial recombinases (RecA,
RecG, RuvC) and human recombinases (Rad51, Rad52
DMC1) were amplified by polymerase chain reaction
(PCR). The PCR product were cloned in pGEM®-T Easy
vector (Promega).
• In vitro expression:- Polyprotein constructs were
used with wheat germ transcription– translation
system (TNT®, Promega) in the presence of [35S]-
Methionine. Radiolabelled protein products were
separated in 10% SDS–PAGE and detected by
autoradiography.
• Plant expression vector pGSC:- All bacterial and

31. a.The coding sequences of bacterial (RecA, RecG and
RuvC) and human (Rad51, Rad52 and DMC1)
Recombinases.
b.The transgene used as ICR substrate in the tobacco
transgenic line N1DC4 is formed of two defective overlapping
fragments of β-glucuronidase (GUS)

32. SDS-PAGE of in vitro TNT
products

33. • Plant transformation:- The constructs in pGSC
binary vector were transferred into Agrobacterium
tumefaciens strain LBA4404. Agrobacterium clones
were used to transform tobacco seedlings of N1DC4
line. The transgenic lines were selected and
transferred to the glasshouse. At maturity pollen and
seeds of the obtained primary transformants were
collected.
• Transgene segregation and seed scoring:- T1
seeds were plated on MS medium in the presence of
100 mg/l of sulphonamide and scored for resistance.
The lines showing 3:1 segregation ratio were selected
to make homozygotes. Homozygous lines and N1DC4
control were grown in the glasshouse and their pollen
CONT…

34. • Intrachromosomal recombination (ICR) assay:- N1DC4 is a
homozygous line containing β-glucuronidase (GUS) based
transgene as a substrate for Intrachromosomal
recombination (ICR). The transgene is formed of two
defective overlapping GUS fragments in direct orientation
and separated by hygromycin resistance gene (hpt). ICR
restores a functional GUS gene that can be detected by
histochemical staining as blue spots on seedlings and blue
pollen. To determine the number of ICR events in somatic
cells, six-week-old seedlings were stained for GUS activity
and the number of blue spots recorded under a binocular
microscope. To monitor ICR in pollen, dehiscent anthers of
three flowers were combined in 1.5 ml microfuge tube
containing 1ml of GUS staining buffer supplemented with
20% methanol to inhibit endogenous GUS activity. The pollen
CONT…

35. ICR restore a functional GUS gene that can be detected by
histochemical staining as blue spots on seedlings (left) and
blue pollen (right)

36. RESULTS
• They expressed several bacterial and human
recombinases in different combinations in a
tobacco transgenic line containing a highly
sensitive β-glucuronidase (GUS)-based ICR
substrate.
• Coordinated simultaneous expression of
multiple recombinases was achieved using the
viral 2A translational recoding system.
• They found that most recombinases increased
ICR dramatically in pollen, where HR will be

37. • DMC1 expression produced the greatest
stimulation of ICR in primary transformants,
with one plant showing a 1000-fold increase
in ICR frequency.
• Evaluation of ICR in homozygous T2 plant lines
revealed increases in ICR of between 2-fold
and 380-fold depending on recombinase(s)
expressed.
• By comparison, ICR was only moderately
CONT…

38. CONCLUSION
S
Opportunities for CRISPR/Cas deployment in plant
biotechnology are currently limited to gene editing
applications but would be greatly expanded by the
addition of full gene replacement (gene targeting)
technology.
Here they show that expression of several bacterial
or eukaryotic recombinases or combinations of
recombinases can dramatically increase ICR in
tobacco.
Greatest increases were seen with the single
recombinases DMC1, RecG and Rad51.
If these stimulations of HR translate to full gene
targeting assays where the efficiency of CRISPR/Cas
is also deployed to generate targeted double strand

39. THANK YOU