Organellar genomes, such as those found in mitochondria and chloroplasts, can be manipulated. The mitochondria genome is maternally inherited and contains genes that code for proteins involved in respiration. The chloroplast genome is also maternally inherited and contains genes for photosynthesis-related proteins. Methods to transform these genomes include particle bombardment, PEG-mediated transformation, and Agrobacterium-mediated transformation. Manipulating organellar genomes has applications for crop improvement like developing cytoplasmic male sterility.

1. ORGANELLAR GENOME AND THEIR MANIPULATION
PRESENTED BY,
Sandesh,G.M
2016610811

2. WHAT IS ORGANELLAR GENOME..?
◦ ORGANELLE :
Mitochondria , Chloroplast, Golgicomplex, Endoplasmic reticulum etc..,
◦ The genome present in the Chloroplast and Mitochondria are called as
Organellar Genome.

3.  Mitochondria (animals and plants)
 Chloroplasts (plants)
1. Mitochondria and chloroplasts occur outside the nucleus, in the
cytoplasm of the cell.
2. Contain genomes (mtDNA/cpDNA) and genes, i.e.,
extrachromosomal genes, cytoplasmic genes, organelle genes,
or extranuclear genes.
3. Inheritance is non-Mendelian (e.g., cytoplasm typically is
inherited from the mother).

4. Endosymbiotic bacteria = Free-living prokaryotes that invaded
ancestral eukaryotic cells and established a mutually beneficial
relationship.
1. Mitochondria - Derived from a photosynthetic purple bacterium
that entered a eukaryotic cell > billion years ago.
2. Chloroplasts - Derived from a photosynthetic cyanobacterium.

5. >1.5 billion years
?
Endosymbiotic theory

6.  Alpha proteobacterial endosymbiont became specialized for respiration.
Mitochondria
 Later - cyanobacterial endosymbiont specialized for photosynthesis in the plant
lineage
Chloroplasts

7.  Small but essential.
Mitochondria (site of respiration).
Plastids (site of photosynthesis) .
 Multiple organelles and organelle genomes per cell.
20 – 20,000 genomes per cell, depending on cell type.
 Organized in nucleoids.
Nucleoprotein complexes containing multiple genome copies.
Not to be confused with nucleosomes.
 Non-Mendelian inheritance.
Commonly maternal.
“Paternal leakage” , especially with interspecific crosses.
Some organisms paternal or bi-parental.

8.  Necessary but insufficient for organelle function.
Support organelle functions.
Membrane-associated respiratory or photosynthetic proteins.
 Support organelle gene expression.
rRNAs , tRNAs and ribosomal proteins.
 Nuclear gene products also required.
Translated in cytosol.
Imported into the organelle.
10% of nuclear genes predicted mitochondrial targeting.
15% of nuclear genes predicted plastid targeting.

9. Wheat
mtDNA
452,528 bp

10.  mtDNA contains genes for:
•tRNAs
•rRNAs
•cytochrome oxidase, NADH-dehydrogenase, & ATPase subunits.
•mtDNA genes occur on both strands.
•Mitochondria’s genetic information also occurs in the nuclear DNA:
•DNA polymerase, replication factors
•RNA polymerase, transcription factors
•ribosomal proteins, translation factors, aa-tRNA synthetase
•Additional cytochrome oxidase, NADH, ATPase subunits.

11. •Most required mitochondrial (and
chloroplast) proteins are coded by nuclear
genes in the nuclear genome.
•Five mtDNA complexes with 13 mtDNA
subunit genes are paired with 76 nuclear
subunit genes to make the same proteins.
I – NADH; II - Succinate dehydrogenase; III -
Cytochrome bc
IV - Cytochrome c oxidase; V - ATP synthase

12. • Replication is semi-conservative (like nuclear DNA replication) and uses DNA
polymerases specific to the mitochondria.
• Occurs throughout the cell-cycle (not just S phase); mitochondria are constantly
created.
• Control region (non-coding) similar to Ori sequence in E. coli forms a
displacement loop (d-loop) that functions in mtDNA replication.
• Mitochondria (organelle) are not synthesized de novo, but grow and divide like
other cells (e.g., mitosis).

14. • mRNAs from the mtDNA are synthesized and translated in the mitochondria.
• Gene products encoded by nuclear genes are transported from the cytoplasm to
the mitochondria.
• Mammalian and other vertebrate mtDNAs are transcribed as a single large RNA
molecule (polycistronic) and cleaved to produce mRNAs, tRNAs, and rRNAs
before they are processed.
• Most mtDNA genes are separated by tRNAs that signal transcription termination.

15. • Mitochondrial mRNAs do not have a 5’ cap (yeast and plant mt mRNAs have a
leader).
• mtDNA-specific initiation factors (IFs), elongation factors (EFs), and release
factors (RFs) are used for translation.
• AUG is the start codon (binds with fMet-tRNA like bacteria).
• Only plants use the “universal” genetic code. Codes for mammals, birds, and
other organisms differ slightly.

16. • Easy to isolate and PCR (high copy #).
• Most mtDNA is inherited maternally. Can be used to assess maternal population
structure (to the exclusion of male-mediated gene flow), Because it is “haploid”
effective population size of mtDNA is 1/4 that of a nuclear gene.
• mtDNA sequences are refered as “haplotypes” not “alleles”
• As a result of drift, mtDNA substitutions “fix” rapidly (due to genetic drift) and
typically show higher levels of genetic differentiation between populations.
Useful for:
• Maternity & forensics (maternal ID)
• Phylogenetic systematics
• Population &conservation genetics)

19. • Leber’s hereditary optic neuropathy (LHON):
• Mid-life adult blindness from optic nerve degeneration.
• Mutations in ND1, ND2, ND4, ND5, ND6, cyt b, CO I, CO II, and ATPase 6 inhibit electron
transport chain.
• Kearns-Sayre Syndrome :
• Paralysis of eye muscles, accumulation of pigment and degeneration of the retina, and
heart disease.
• Deletion of mtDNA tRNAs.
• Myoclonic epilepsy & ragged-red fiber disease (MERRF) :
• Spasms and abnormal tissues, accumulation of lactic acid in the blood, and uncoordinated
movement.
• Nucleotide substitution in the mtDNA lysine tRNA.
Most individuals with mtDNA disorders possess a mix of normal and mutant
mtDNA, therefore severity of diseases varies depending on the level of normal
mtDNA.

22.  Chloroplast organelles are the site of photosynthesis and occur only in green plants
and photosynthetic protists,
 Like mtDNA, chloroplast genome is:
• Circular, double-stranded
• Lacks structural proteins
• %GC content differs
 Chloroplast genome is much larger than animal mtDNA, ~80-600 kb.
 Chloroplast genomes occur in multiple copies and carry lots of non-coding DNA.
 Complete chloroplast sequences have been determined for several organisms
(tobacco 155,844 bp; rice 134,525 bp).

24.  Nuclear genome encodes some chloroplast components, and cpDNA codes the rest,
including:
 2 copies of each chloroplast rRNA (16S, 23S, 4.5s, 5S)
 tRNAs (30 in tobacco and rice, 32 in liverwort)
 100 highly conserved ORFs (~60 code for proteins required for transcription,
translation, and photosynthesis).
 Genes are coded on both strands (like mtDNA).
 cpDNA translation- similar to prokaryotes:
1. Initiation uses fMet-tRNA.
2. Chloroplast specific IFs, EFs, and RFs.
3. Universal genetic code.

25. • Ratios typical of Mendelian segregation do not
occur because meiotic segregation is not involved.
• Reciprocal crosses usually show uniparental
inheritance because zygotes typically receive
cytoplasm only from the mother.
• Genotype and phenotype of offspring is same as
mother.

26. Copies of mtDNA and chloroplast genes can be transposed to
the nuclear genome and vice versa.
Mitochondrion Chloroplast
Nucleus

27. Organellar genome manipulation
Plant cells is well equipped with three types of genomes, viz., nuclear,
mitochondrial and chloroplast.
Among these genomes, nuclear genes are inherited by both the parents.
Whereas chloroplast and mitochondrial genes are inherited maternally,
therefore chloroplast and mitochondrial genomes could act as a useful
candidates for transgene containment which is a crucial concern in genetically
modified crops, where transgene escape is a major concern.
(Siddra Ijaz 2010).

28. Mitochondrial genome manipulation
mtDNA mutations in humans cause severe neurodegenerative diseases that
are currently incurable and await the development of gene therapy strategies.
In plants, mitochondrial genome rearrangements are the basis of cytoplasmic
male sterility (CMS), a key genetic tool for the production of hybrids in
breeding protocols.
The product of the transgene is compartmentalized, opening the possibility
to express proteins otherwise toxic for the cell.
Due to the maternal inheritance of mitochondria in most species, the
transgene will not be spread through the pollen.

29. Contd..,
Plant mitochondria offer great advantages in its manipulation because of
following reasons.
1. Maternal inheritance.
2. No pleiotropic effect.
3. Absence of gene silencing.
4. Multigene engineering.
5. No position effects.
6. No specific degradation of transgene RNA at post transcriptional level.

30. Methods of mitochondrial transformation
Protoplast fusion.
Particle bombardment of cell culture.
 Agrobacterium mediated gene transfer.
 Microinjection method.

31. Contd..,

32. 1. Electroporation:
◦ Delivery of Nucleic Acids into Isolated Mitochondria:
◦ One of the first described approaches to transfer DNA into isolated
mitochondria was electroporation.
◦ Relatively large DNA molecules were thus introduced into mouse organelles
(Collombet et al. 1997; Yoon and Koob 2003).
◦ Gene constructs were also successfully electroporated into isolated
trypanosomatid mitochondriaand expressed in organello (Estevez et al. 1999)

33. 2. Conjugation:
◦ An original approach to introduce DNA into the mitochondria was described
as bacteria-to-mitochondria conjugation (Yoon and Koob 2005).
◦ conjugative-competent Escherichia coli cells could transfer a DNA construct
containing an origin of transfer ( oriT) into the matrix of purified mouse
mitochondria.

34. 3. Exploiting the Protein Import Machinery:
◦ The translocation of the nuclear-encoded mitochondrial proteins into the
organelles.
◦ A single-stranded or double-stranded oligonucleotide covalently linked to the
C-terminus of a mitochondrial precursor protein could be introduced into
isolated yeast ( Saccharomyces cerevisiae) mitochondria (Vestweber and
Schatz 1989)

36. Applications of mtDNA transfer
• Many economically important crop species are devoid of CMS system due to
unavailability of cytoplasmic genetic male sterility. In such species
mitochondrial manipulation could provide a novel means to develop CMS lines
as nonGMO/ transgenic materials.
• Once the mitochondrion is transformed with "gene of interest", their
maternal inheritances will confined the gene through successive generations
thus reducing the risk of transgene escape.
• Use of mitochondrial plasmid as a vector for transgene would be more
compatible than the bacterial plasmid being its origin from the plant genome
itself.

37. Chloroplast genome manipulation
Methods of genome manipulation.
1.Biolistic DNA delivery.
2.PEG mediated transformation.
3.Agrobacterium mediated transformation.
4.Microinjection.

38. Contd..,
1. Biolistic DNA delivery :
Genegun is used.
High pressure He gas is used as propellant.
Tungsten or gold particles are used.
Leaves, cotyledons are cultured cells are most suitable.

39. Contd..,
2. PEG mediated transformation:
◦ Exposing of the protoplasts to purified DNA in presence of PEG.
◦ Here regeneration step is required.
◦ Long selection time is required after initial DNA delivery.
◦ Technically demanding and require tissue culture skills.
3. Agrobacterium mediated gene transformation:
◦ Not successful yet.

40. Contd..,4. GALINSTAN EXPANSION FEMTOSYRINGE :
◦ This is a novel approach that involves the microinjection of DNA into chloroplast.
◦ This was given by Knoblauch et.al (1999).
◦ The heat induced expansion of liquid metal Galinstan within the glass syringe forces
the transformation of plastid DNA through a capillary tip with a diameter of
approximately 0.1mm.
Chl autofluroscence GFP fluroscence Overlay of both channels

41. Successful genome manipulation in chloroplast

42. Advantages of cDNA genome manipulation
• Plastid transformation is the ability to accumulate in the chloroplast any
foreign proteins or their biosynthetic products that could be harmful if they
were in the cytoplasm.
Ex: Cholera toxin B subunit (CTB), a candidate oral subunit vaccine for cholera,
was non-toxic when accumulated in large quantities within transgenic plastids
yet was toxic when expressed in leaves via the nuclear genome, even at very
low levels.
• Multigene engineering through the chloroplast genome is possible in a single
transformation event.