r dna technology

1. BY
A.AROCKIA DAISY
MSC BIOTECHNOLOGY

2.  Ti plasmids are tumour- inducing plasmids present in
Agrobacterium tumefaciens.
 They are large, circular, double stranded DNA
molecules acting as extra chromosomal DNAs.
 The size of the ti plasmid ranges from 150-230kbp
 The molecular weight of the plasmids is 120-160mega
daltons
 They are transferred from Agrobactrium to the plant
cell during infection.

3. The Ti plasmids has a,
T-DNA segments
Two border sequences,
Tragene
A sequence for opine catabolism,
An incompatibility region,
Origin of replication
Vir genes

4.  The Ti plasmids are divided into two types
based on the type of opine they synthesis.
 They are,
Octopine Ti plasmids
 Nopaline Ti plasmids
 All these Ti plasmids are identical in structure
except in their sequences foe opine synthesis
and opine catabolism.

5.  Octopine Ti plasmids encode for the synthesis of the
opine,octopine.
 Octopine is an unusual amino acid synthesised from
arginine and pyruvate by condensation.
 The molecular formula of octopine is C9H18N4O4.
 The plant cell infected by Agrobacterium that has
octopine Ti plasmid has high concentration of octopine.
 Ti plasmid of Agrobacterium tumefaciens Ach5 and
Agrobacterium tumefaciens B6 are examples of
octopine Ti plasmid.

7.  This type of Ti plasmids encodes for the
synthesis of Nopaline, anopine from a
ketoglutarate and arginine.
 Ti plasmid of agrobacterium tumefaciens C58 is the
example for nopaline Ti plasmid
 The molecular formula of nopaline C9H20N4O6.
 The pTi C58 is 1,94,000 basepairs in size.

9.  Ri Plasmids of root inducing plasmids
 They are circular double stranded DNAs found in the
soil bacterium agrobacterium rhizogens.
 They are analogous to Ti plasmids of a agrobacterium
tumefaciens.
 They can transfer asegment of their DNA to the
chromosome of the plant cell.
 Hence Ri plasmids are used to construct gene cloning
vectors for dicot plants.

10.  Ri plasmid is a large plasmid with the size of 250 kbp.
 Ri plasmid has two DNAs.
 The right T-DNA has genes homologous to those of T-
DNA of Ti plasmids. They include tms1 and tms2 with
two border sequences.
 The left T-DNA is about 21kbp long and is entirely
different from Ti plasmid. It has four genes
rolA,rolB,rolC and rolD

12.  Ri plasmids can be used as substitutes for Ti
plasmids to transfer forgien genes to plants
 Ri plasmids exchange DNA segments between
plants and bacteria and hence they could be
used to study gene exchange between the
organisms.
 They could be used to induce rooting in clones
where rooting from callus or explant is difficult.
 They could also be used to increase nodulation
frequency of plantlets during micropropagation.

13.  Animal viruses capable of transmitting foreign genes to
animal cells are called viral vectors.
 The following viruses can be used in gene cloning in
animal cells.
 Simian virus 40
Bovine papilloma virus
Baculovirus
Vaccinia virus
Adenovirus
Retroviruses

14.  SV40 is a small virus that infects monkeys.
 It causes cytoplasmic vacuolation in infected cells and
hence it is known as simian vacuolating virus 40.
 It is included under the group papova viruses.
 SV40 is an oncogenic virus.
 The DNA of SV40 contains an origin of replication,a
set of genes called early genes and another set of genes
called late genes.

16.  Baculo viruses infect many insects.
 Their capsid is rod-shaped and the genome is a circular
double stranded DNA.
 The virus particles remain embedded in a matrix
protein called polyhedrin.
 Baculo viruses such as Autographa California nuclear
polyhedrosis virus and Bombyx Mori nuclear
polyhedrosis virus are used as gene cloning vectors for
cultured animal cells.

17.  Researchs have confirmed that expression of
polyhedrin gene is derived by an extremely strong
promoter and that polyhedrin gene is not essential for
virus replication.
 Since we cannot use 130kb long Baculovirus DNA, the
polyhedrin gene is advantages to construction gene
transfer vectors.

19.  Retro viruses single stranded RNA virusesinfecting
birds, mammels and other animals.
 They are icosahedral viruses with 100nm diameter.
 They are enveloped viruses. The RNAsynthesized
double stranded DNA by means of reverse
transcription.

20.  These features of Retro viruses give impetous to
develop gene transfer systems for gene cloning in
animal cells.
 Example ; Murine leukaemia virus.

22. THAN
K
YOU