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CLONING VECTORS FOR HIGHER
PLANTS AND ANIMALS
SOUSAN
A274153121006
HG&MM
TOBACCO MOSAIC VIRUS
➢RNA virus
➢Designing expression vector
➢TMV was the first virus to be purified and sequenced
➢The single stranded RNA genetic material of TMV encloses 6300
nucleotides with four open reading frames
INTRODUCTION
➢A plant virus
➢Genus Tobamovirus
➢Infects tobacco plants, potatoes, tomatoes,
and other members of the Solanaceae
family
➢Creates a mosaic-like pattern, mottling,
and discoloration of the leaves
STRUCTURE
➢The tobacco mosaic virus (TMV) has a rod-like appearance that is 300 nm
long with a diameter of 18 nm
➢It is covered by a protein shell called capsid that encloses the virus’s genetic
material
➢The genetic material is a single-stranded RNA molecule
➢The capsid is made up of 2130 molecules of coat proteins that assemble in a
rod-like helical structure possessing 16.3 proteins per helix turn
➢The RNA is found in a coiled manner inside the capsid coat and is made up
of approximately 6395 nucleotides
➢It has structural chirality and inherent symmetry in the structure which
gives the organism an easy way for chemical or genetic modifications
LIFE CYCLE
➢The virus gets inside the plant host cells via vectors such as aphids, flies
and other insects
➢After entering the host cell the virus starts to multiply and spreads to the
nearby cells via plasmodesmata
➢The virus can be transmitted to other healthy plants by coming in direct
contact with the infected plants
➢For replication and multiplication inside the host cell, the viral particle
releases its genomic material
➢The ssRNA then translates multiple mRNAs that can replicate the viral
genome and the capsid proteins
➢All these proteins assemble to form a newly synthesised cell of tobacco
mosaic virus that is ready to infect another
PROTEIN ENCODED BY TMV
➢Tobacco mosaic virus (TMV) encodes four known functional proteins:
the 126 and 183 kDa replication-associated proteins, the movement
protein (MP), and the structural capsid or coat protein (CP)
➢In order to have a successful infection, these four multifunctional
proteins cooperate with many host components
➢The 17.5 kDa coat protein (CP) of TMV is a multifunctional protein
responsible for virion formation, viral systemic movement, cross-
protection and genomic RNA stability
MECHANISM OF TMV
AGROBACTERIUM TUMIFACIENS
➢Ti-plasmid, short for tumour-inducing plasmid, is an
extrachromosomal molecule of DNA found commonly in the plant
pathogen Agrobacterium tumefaciens
➢Agrobacterium is a gram negative bacteria that belongs to the class
Alphaproteobacteria
➢The Ti-plasmid in the bacteria is known to induce crown gall disease
in plants by transferring crucial regions from the plasmid
➢These crucial regions were seen to modify the plant cells into a
tumour to produce synthetic plant hormones and cause crown gall
FEATURES OF TI PLASMID
➢Virulence Region: The virulence region codes for virulence genes that are
responsible for the transfer of T-DNA to the plant cells and also recruiting
various effector proteins for infecting the plant cells
➢T-DNA: The T-DNA region is the crucial region that gets transferred to the
plant cell for infection. It is approximately 15-20 kbp in length and is
transferred to the plant cell via means of genetic recombination
➢Opine Catabolism: The opine catabolism region is the region from where
the bacteria sources its nutrients for the whole process. Opines are
derivatives of amino acid or sugar phosphates that can be catabolized to use
in the form of nutrients. The types of opines found in Ti-plasmid are
nopaline and octopine types
➢Origin of Replication: The origin of replication is the region where
replication of the plasmid is initiated
APPLICATION
➢The ability of the Ti-plasmid to modify the plant cells has been taken
advantage of for the production of transgenic plants
➢The plasmids have been modified into a cloning vector now which are
no more pathogenic to plants. They are being used to transfer genes of
our interest into the plant and produce plants with better quality and
quantity.
➢It is therefore known as ‘nature’s genetic engineer’
AGROBACTERIUM-MEDIATED GENE
TRANSFER (TRANSFORMATION) IN PLANTS
➢The basis of Agrobacterium-mediated transformation is the ability of the organism
to transfer its T-DNA into the host cells efficiently
➢The biology of the process consists of two components; the T-DNA consists of 25
bp repeats that end at the T-region and the virulence (vir) region composed of
seven major loci
➢The mechanism of Agrobacterium-mediated transformation is based on the
transfer of a piece of plasmid by the bacteria into the plant cells during infection
➢The plasmid then integrates into the nuclear genome in order to express its own
genes and affect the hormonal balance in the host cell
➢Besides, the bacteria also produce a number of enzymes that are involved in the
synthesis of opines that is then used by the bacteria as nutrients
➢Some of the essential components of the bacteria involved in infection are T-DNA
present on the plasmid called Ti (tumor-inducing) plasmid along with other
functional components like virulence (vir), conjugation (con), and origin
of replication (ori)
➢The infection begins with the entry of the bacteria through wounded sites. The binding of
bacteria to the plant cells is enhanced by the release of phenolic acetosyringone (AS) by the
injured plant cells
➢The AS activates the VirA proteins on the bacteria, which activates VirG via phosphorylation
of its aspartate residue
➢The activated form of VirG then binds to other vir genes, inducing their expression. VirD
activated by this process stimulates the T-strand generation (a single-stranded copy of the T-
DNA)
➢The VirD2 covalently binds to the 5’ end of the T-strand as the 5’ end is the leading end during
the transfer. Other factors like VirE2 and VirB proteins also bind to the T-strand, forming a T-
complex
➢The complex is then passed into the nucleus by the nuclear target signals released by the
Vir proteins. The T-DNA strand is integrated into the plant genome randomly as either a
single copy or multiple copies
➢The integration usually occurs in the transcription active or repetitive regions of the
genome by the process of recombination
➢Even though much is known about the molecular biology of T-DNA transfer
in Agrobacterium cells, not much is known about the plant-encoded factors involved in
the process
PROTEINS ENCODED
➢The ability of Agrobacterium to transform plants and other organisms is
under highly regulated genetic control
➢Two Virulence (Vir) proteins, VirA and VirG, function as a two-component
regulatory system to sense particular phenolic compounds synthesized by
wounded plant tissues
➢Ti plasmid-encoded genes responsible for the catabolism of the crown gall
opine mannopine by Agrobacterium tumefaciens are homologs of the T-
region genes responsible for the synthesis of this opine by the plant tumor
APPLICATIONS
➢The Agrobacterium-mediated transformation has been used as a method of genetic modification of plants for
the production of various substances like proteins, antibodies, and even vaccines
➢Different plants have also been modified to produce life-saving pharmaceutical products like anticoagulants,
human epidermal growth factors, and interferons
➢Transgenic plants prepared with Agrobacterium serve as biomonitors to detect the presence of toxic
compounds in the environment as well as to detoxify the contaminated soil and water
➢Agrobacterium-mediated transformation has also remarkably increased crop yields by modifying the shelf-life
and biosynthesis of the plants
➢Plants can be modified to enhance tolerance against biotic and abiotic factors, nutrient capture with increased
pest resistance
➢Agrobacterium-mediated transformation has been used to produce insect resistance crops by the incorporation
of various toxic genes like the Bt toxin genes
➢The increase in pest resistance results in a reduction in the use of harmful agrochemicals and herbicides
➢Agrobacterium-mediated transformation is one of the less complicated genetic engineering techniques which
has the possibility of being upgraded to use with other organisms as well
SV 40
➢Simian virus 40
➢Belongs to polyoma viruses group
➢The virus was first isolated as contaminant from a monkey kidney
culture used for the production of poliovirus vaccine
➢Its genome is very small and can be easily modified for gene therapy
purpose
➢Its genome has ds circular DNA wrapped up as nucleosome with the
help of histone proteins
➢It is roughly 40-45 nm in diameter
➢The chromosome is 5000 bp long. The genome of SV40 is like a mini
chromosome
➢The genome of SV40 consists of early proteins, late proteins, and
regulatory proteins
➢Early proteins are non-structural while late proteins are structural
proteins
➢Regulatory region consists of promoter, enhancer, and origin of
replication
➢The capsid proteins are produced later
which then forms viable virus and
comes out of the cell
➢The early protein-coding genes can be
replaced by the gene of interest. The
mutation in the p53 promoter which
leads to cancerous conditions was
discovered in the SV40 virus
➢Gene therapy using recombinant SV40
is a highly suitable means to transfer
the gene in a number of conditions
➢Recombinant SV40 viral vectors are made in some plasmid
backgrounds such as pGEMT plasmid vector
➢The permissible cells are transfected with the plasmid along with the
support plasmid needed to package the viral genome
➢The recombinant virus containing the transgene is then recovered by
the lysis of the transfected cell
➢The late proteins of SV40 are synthesized from the opposite strand.
Out of four structural proteins, VP1 is the most abundant one
➢The minimum viral-specific sequence required to assemble the virion
consists of the origin of replication and encapsulation sequences they
were overlaps with the early promoter region in the genome
➢The transcription of the genome and the transgene is governed by the
pol 3 promoter
LIFE CYCLE OF SV40
MECHANISM OF SV40
FEATURES OF A SV40 BASED VECTOR
➢Capacity of the transgene is limited to 5Kb
➢Both resting, as well as dividing cells, are equally transduced by
SV40-based vectors
➢High level of transduction efficiency
➢ Long-lasting transgene expression. It has been reported that the
transgene expression once established will remain lifelong
➢Neutralizing antibody against the recombinant antigen
ADENOVIRUS
➢Adenoviruses can be converted into efficient gene transfer vehicles
➢Adenoviral vectors are not inherently dangerous
➢The dose of vector delivered is related to the toxicity observed
➢Icosahedral shape
➢Composed of 13% DNA & 87% protein
➢ Non-enveloped viruses with an icosahedral nucleocapsid containing a
double stranded DNA genome
➢Size 90-100 nm
➢Since humans commonly come in contact with adenoviruses which
cause respiratory, gastrointestinal, and eye infections, the majority of
patients have already developed neutralizing antibodies which can
inactivate the virus before it can reach the target cell
➢To overcome this problem scientists are currently investigating
adenoviruses that infect different species to which humans do not have
immunity
➢Their primary applications are in gene therapy and vaccination
ADENOVIRAL GENOME
➢The adenoviral genome is a linear , 36 Kb double-stranded DNA
➢It can take up 8Kb foreign DNA
PROTEINS ENCODED FOR ADENOVIRUS
➢The adenovirus genome is coded with at least 50 proteins
➢In adenovirus serotype 2, 6 of these proteins (pVI, pVII, pVIII, pTP, pllla,
and L2 11 kDa) are synthesized as precursors, which are processed late in
infection
➢The adenovirus (Ad) capsid protects the viral DNA and acts as a vessel for
the delivery of the genetic material to the nucleus of the infected cell.
➢ For human Ad serotype 5 (Ad5), the capsid contains three major
proteins, hexon, penton, and fiber, and five minor proteins, VI, VIII, IX,
IIIa, and IVa2
➢The outer shell of the adenovirus (Ad) capsid comprises three major types
of proteins: hexon, penton base, and fiber
APPLICATIONS
➢Viral vectors were originally developed as an alternative to
transfection of naked DNA for a molecular genetics experiment
➢Protein coding genes can be expressed using viral vectors commonly
to study the function of the particular protein
➢Viral vectors, especially retroviruses, stably expressing marker genes
are widely used to permanently label cells to track them and their
progeny
➢It is used in gene therapy
➢ It is used in the production of vaccines
THANK YOU

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Cloning vectors for higher plants and animals

  • 1. CLONING VECTORS FOR HIGHER PLANTS AND ANIMALS SOUSAN A274153121006 HG&MM
  • 2. TOBACCO MOSAIC VIRUS ➢RNA virus ➢Designing expression vector ➢TMV was the first virus to be purified and sequenced ➢The single stranded RNA genetic material of TMV encloses 6300 nucleotides with four open reading frames
  • 3. INTRODUCTION ➢A plant virus ➢Genus Tobamovirus ➢Infects tobacco plants, potatoes, tomatoes, and other members of the Solanaceae family ➢Creates a mosaic-like pattern, mottling, and discoloration of the leaves
  • 4. STRUCTURE ➢The tobacco mosaic virus (TMV) has a rod-like appearance that is 300 nm long with a diameter of 18 nm ➢It is covered by a protein shell called capsid that encloses the virus’s genetic material ➢The genetic material is a single-stranded RNA molecule ➢The capsid is made up of 2130 molecules of coat proteins that assemble in a rod-like helical structure possessing 16.3 proteins per helix turn ➢The RNA is found in a coiled manner inside the capsid coat and is made up of approximately 6395 nucleotides ➢It has structural chirality and inherent symmetry in the structure which gives the organism an easy way for chemical or genetic modifications
  • 5.
  • 6. LIFE CYCLE ➢The virus gets inside the plant host cells via vectors such as aphids, flies and other insects ➢After entering the host cell the virus starts to multiply and spreads to the nearby cells via plasmodesmata ➢The virus can be transmitted to other healthy plants by coming in direct contact with the infected plants ➢For replication and multiplication inside the host cell, the viral particle releases its genomic material ➢The ssRNA then translates multiple mRNAs that can replicate the viral genome and the capsid proteins ➢All these proteins assemble to form a newly synthesised cell of tobacco mosaic virus that is ready to infect another
  • 7.
  • 8. PROTEIN ENCODED BY TMV ➢Tobacco mosaic virus (TMV) encodes four known functional proteins: the 126 and 183 kDa replication-associated proteins, the movement protein (MP), and the structural capsid or coat protein (CP) ➢In order to have a successful infection, these four multifunctional proteins cooperate with many host components ➢The 17.5 kDa coat protein (CP) of TMV is a multifunctional protein responsible for virion formation, viral systemic movement, cross- protection and genomic RNA stability
  • 10. AGROBACTERIUM TUMIFACIENS ➢Ti-plasmid, short for tumour-inducing plasmid, is an extrachromosomal molecule of DNA found commonly in the plant pathogen Agrobacterium tumefaciens ➢Agrobacterium is a gram negative bacteria that belongs to the class Alphaproteobacteria ➢The Ti-plasmid in the bacteria is known to induce crown gall disease in plants by transferring crucial regions from the plasmid ➢These crucial regions were seen to modify the plant cells into a tumour to produce synthetic plant hormones and cause crown gall
  • 11.
  • 12. FEATURES OF TI PLASMID ➢Virulence Region: The virulence region codes for virulence genes that are responsible for the transfer of T-DNA to the plant cells and also recruiting various effector proteins for infecting the plant cells ➢T-DNA: The T-DNA region is the crucial region that gets transferred to the plant cell for infection. It is approximately 15-20 kbp in length and is transferred to the plant cell via means of genetic recombination ➢Opine Catabolism: The opine catabolism region is the region from where the bacteria sources its nutrients for the whole process. Opines are derivatives of amino acid or sugar phosphates that can be catabolized to use in the form of nutrients. The types of opines found in Ti-plasmid are nopaline and octopine types ➢Origin of Replication: The origin of replication is the region where replication of the plasmid is initiated
  • 13. APPLICATION ➢The ability of the Ti-plasmid to modify the plant cells has been taken advantage of for the production of transgenic plants ➢The plasmids have been modified into a cloning vector now which are no more pathogenic to plants. They are being used to transfer genes of our interest into the plant and produce plants with better quality and quantity. ➢It is therefore known as ‘nature’s genetic engineer’
  • 14.
  • 15. AGROBACTERIUM-MEDIATED GENE TRANSFER (TRANSFORMATION) IN PLANTS ➢The basis of Agrobacterium-mediated transformation is the ability of the organism to transfer its T-DNA into the host cells efficiently ➢The biology of the process consists of two components; the T-DNA consists of 25 bp repeats that end at the T-region and the virulence (vir) region composed of seven major loci ➢The mechanism of Agrobacterium-mediated transformation is based on the transfer of a piece of plasmid by the bacteria into the plant cells during infection ➢The plasmid then integrates into the nuclear genome in order to express its own genes and affect the hormonal balance in the host cell ➢Besides, the bacteria also produce a number of enzymes that are involved in the synthesis of opines that is then used by the bacteria as nutrients ➢Some of the essential components of the bacteria involved in infection are T-DNA present on the plasmid called Ti (tumor-inducing) plasmid along with other functional components like virulence (vir), conjugation (con), and origin of replication (ori)
  • 16. ➢The infection begins with the entry of the bacteria through wounded sites. The binding of bacteria to the plant cells is enhanced by the release of phenolic acetosyringone (AS) by the injured plant cells ➢The AS activates the VirA proteins on the bacteria, which activates VirG via phosphorylation of its aspartate residue ➢The activated form of VirG then binds to other vir genes, inducing their expression. VirD activated by this process stimulates the T-strand generation (a single-stranded copy of the T- DNA) ➢The VirD2 covalently binds to the 5’ end of the T-strand as the 5’ end is the leading end during the transfer. Other factors like VirE2 and VirB proteins also bind to the T-strand, forming a T- complex
  • 17. ➢The complex is then passed into the nucleus by the nuclear target signals released by the Vir proteins. The T-DNA strand is integrated into the plant genome randomly as either a single copy or multiple copies ➢The integration usually occurs in the transcription active or repetitive regions of the genome by the process of recombination ➢Even though much is known about the molecular biology of T-DNA transfer in Agrobacterium cells, not much is known about the plant-encoded factors involved in the process
  • 18.
  • 19. PROTEINS ENCODED ➢The ability of Agrobacterium to transform plants and other organisms is under highly regulated genetic control ➢Two Virulence (Vir) proteins, VirA and VirG, function as a two-component regulatory system to sense particular phenolic compounds synthesized by wounded plant tissues ➢Ti plasmid-encoded genes responsible for the catabolism of the crown gall opine mannopine by Agrobacterium tumefaciens are homologs of the T- region genes responsible for the synthesis of this opine by the plant tumor
  • 20. APPLICATIONS ➢The Agrobacterium-mediated transformation has been used as a method of genetic modification of plants for the production of various substances like proteins, antibodies, and even vaccines ➢Different plants have also been modified to produce life-saving pharmaceutical products like anticoagulants, human epidermal growth factors, and interferons ➢Transgenic plants prepared with Agrobacterium serve as biomonitors to detect the presence of toxic compounds in the environment as well as to detoxify the contaminated soil and water ➢Agrobacterium-mediated transformation has also remarkably increased crop yields by modifying the shelf-life and biosynthesis of the plants ➢Plants can be modified to enhance tolerance against biotic and abiotic factors, nutrient capture with increased pest resistance ➢Agrobacterium-mediated transformation has been used to produce insect resistance crops by the incorporation of various toxic genes like the Bt toxin genes ➢The increase in pest resistance results in a reduction in the use of harmful agrochemicals and herbicides ➢Agrobacterium-mediated transformation is one of the less complicated genetic engineering techniques which has the possibility of being upgraded to use with other organisms as well
  • 21. SV 40 ➢Simian virus 40 ➢Belongs to polyoma viruses group ➢The virus was first isolated as contaminant from a monkey kidney culture used for the production of poliovirus vaccine ➢Its genome is very small and can be easily modified for gene therapy purpose ➢Its genome has ds circular DNA wrapped up as nucleosome with the help of histone proteins
  • 22. ➢It is roughly 40-45 nm in diameter ➢The chromosome is 5000 bp long. The genome of SV40 is like a mini chromosome ➢The genome of SV40 consists of early proteins, late proteins, and regulatory proteins ➢Early proteins are non-structural while late proteins are structural proteins ➢Regulatory region consists of promoter, enhancer, and origin of replication
  • 23. ➢The capsid proteins are produced later which then forms viable virus and comes out of the cell ➢The early protein-coding genes can be replaced by the gene of interest. The mutation in the p53 promoter which leads to cancerous conditions was discovered in the SV40 virus ➢Gene therapy using recombinant SV40 is a highly suitable means to transfer the gene in a number of conditions
  • 24. ➢Recombinant SV40 viral vectors are made in some plasmid backgrounds such as pGEMT plasmid vector ➢The permissible cells are transfected with the plasmid along with the support plasmid needed to package the viral genome ➢The recombinant virus containing the transgene is then recovered by the lysis of the transfected cell
  • 25. ➢The late proteins of SV40 are synthesized from the opposite strand. Out of four structural proteins, VP1 is the most abundant one ➢The minimum viral-specific sequence required to assemble the virion consists of the origin of replication and encapsulation sequences they were overlaps with the early promoter region in the genome ➢The transcription of the genome and the transgene is governed by the pol 3 promoter
  • 26.
  • 29. FEATURES OF A SV40 BASED VECTOR ➢Capacity of the transgene is limited to 5Kb ➢Both resting, as well as dividing cells, are equally transduced by SV40-based vectors ➢High level of transduction efficiency ➢ Long-lasting transgene expression. It has been reported that the transgene expression once established will remain lifelong ➢Neutralizing antibody against the recombinant antigen
  • 30. ADENOVIRUS ➢Adenoviruses can be converted into efficient gene transfer vehicles ➢Adenoviral vectors are not inherently dangerous ➢The dose of vector delivered is related to the toxicity observed
  • 31. ➢Icosahedral shape ➢Composed of 13% DNA & 87% protein ➢ Non-enveloped viruses with an icosahedral nucleocapsid containing a double stranded DNA genome ➢Size 90-100 nm
  • 32. ➢Since humans commonly come in contact with adenoviruses which cause respiratory, gastrointestinal, and eye infections, the majority of patients have already developed neutralizing antibodies which can inactivate the virus before it can reach the target cell ➢To overcome this problem scientists are currently investigating adenoviruses that infect different species to which humans do not have immunity ➢Their primary applications are in gene therapy and vaccination
  • 33. ADENOVIRAL GENOME ➢The adenoviral genome is a linear , 36 Kb double-stranded DNA ➢It can take up 8Kb foreign DNA
  • 34.
  • 35. PROTEINS ENCODED FOR ADENOVIRUS ➢The adenovirus genome is coded with at least 50 proteins ➢In adenovirus serotype 2, 6 of these proteins (pVI, pVII, pVIII, pTP, pllla, and L2 11 kDa) are synthesized as precursors, which are processed late in infection ➢The adenovirus (Ad) capsid protects the viral DNA and acts as a vessel for the delivery of the genetic material to the nucleus of the infected cell. ➢ For human Ad serotype 5 (Ad5), the capsid contains three major proteins, hexon, penton, and fiber, and five minor proteins, VI, VIII, IX, IIIa, and IVa2 ➢The outer shell of the adenovirus (Ad) capsid comprises three major types of proteins: hexon, penton base, and fiber
  • 36.
  • 37. APPLICATIONS ➢Viral vectors were originally developed as an alternative to transfection of naked DNA for a molecular genetics experiment ➢Protein coding genes can be expressed using viral vectors commonly to study the function of the particular protein ➢Viral vectors, especially retroviruses, stably expressing marker genes are widely used to permanently label cells to track them and their progeny ➢It is used in gene therapy ➢ It is used in the production of vaccines
  • 38.