In brief about organellar genome

1. UNIVERSITY OF AGRICULTURAL SCIENCES
DEPARTMENT OF PLANT BIOTECHNOLOGY
COURSE : PBT 502 (3+0) FUNDAMENTALS OF MOLECULAR BIOLOGY
SUBMITTED TO : Dr. K.M. Harini Kumar
Professor
Dept. Plant biotechnology

2. Organellar genome and its
composition
Presented by :
SHILPA C
PALB9313

3.  What is organelle ?
An organelle is a specialized subunit within a cell that
perform specific function.
 What is genome ?
A genome is the genetic material of an organism. It consists
of DNA / RNA. The genome includes both the genes , which
includes coding regions and noncoding regions.

4. What is organellar genome ?
Genome present in organelle which include chloroplast and mitochondria.
It is also called as extrachromosomal genome, cytoplasmic genome , extra
nuclear genome.

5. Composition of organellar genome
CONSTITUENTS PERCENT DRY
WEIGHT
Nucleic Acid
RNA
DNA
3-4
< 0.02 – 0.1

6. Endosymbiotic evolution of organellar genome
 Intracellular endosymbionts
that originally descended from
free-living prokaryotes have
been important in the
evolution of eukaryotes by
giving rise to two cytoplasmic
organelles.
 Mitochondria arose from
alpha-proteobacteria and
chloroplasts arose from
cyanobacteria.
 Both organelles have made
substantial contributions to the
complement of genes that are
found in eukaryotic nuclei
today.

8. HISTORY OF ORGANELLAR GENOME

9. INTRODUCTION :
Mitochondria and chloroplast are partly
independent or semi-autonomous as they can
manufacture some of the proteins required for
their functioning with the help of their DNA,
RNAs, enzymes, and ribosomes and obtain the
others from the cytoplasm formed under the
control of the nuclear DNA.

10. • The ancestors of both organelle with genomes encoding around 5,000 genes. During the
course of endosymbiosis, genes are transferred from each organelle to the hosts’ nuclear
genome, and the corresponding gene products are imported back to the organelles.
• The initial genome size of around 5,000 genes decreased to 3–67 genes in mitochondria
and 23–200 genes in chloroplasts.
1.Why these organelle became semi-autonomous ?

12. 2. Why retention of some genes ?
Code disparity hypothesis (CDH)
the genetic code of some mitochondrial
genomes differs from the standard nuclear code,
such that a transferred gene would encode an
incorrect amino acid sequence
especially a codon encoding STOP in the nucleus
but not in the organelle, retains genes in the
organelle if they contain any such codons. This
barrier to functional gene transfer from nucleus
to mitochondria.

14. That the proteins most
frequently encoded in
mitochondria are generally
very hydrophobic, which
may impede their import
after synthesis in the cytosol.
Many nuclear-coded
mitochondrial proteins are
highly hydrophobic,
consequent resistance to
import
Hydrophobicity hypothesis
(HH)

15. Co-location for redox regulation (CORR)
1. That chloroplasts and mitochondria contain those genes whose expression
is required to be under the direct, regulatory control of the redox state of
their gene products, or of electron carriers with which those gene products
interact.
Such genes comprises:
 A core or primary subset of organellar genes. The requirement for
redox control of each gene in the primary subset then confers an
advantage upon location of that gene within the organelle.
 Natural selection therefore anchors some genes in organelles, while
favouring location of others in the cell nucleus.

16. 2.Chloroplast and mitochondrial genomes also contain genes for
components of the chloroplast and mitochondrial genetic systems
themselves.
These genes comprise:
 A secondary subset of organellar genes contains genetic system genes.
There is generally no requirement for redox control of expression of
genetic system genes, though their being subject to redox control.
 Retention of genes of the secondary subset (genetic system genes) is
necessary for the operation of redox control of expression of genes in
the primary subset.
 If all genes disappear from the primary subset, CoRR predicts that there
is no function for genes in the secondary subset, and such organelles
will then, eventually, lose their genomes completely. However, if even
only one gene remains under redox control, then an organelle genetic
system is required for the synthesis of its single gene product.

18. Hypothesis Summary statement of hypothesis
Code disparity hypothesis (CDH) Divergence of genetic code, especially a codon encoding STOP in the nucleus
but not in the organelle, retains genes in the organelle if they contain any
such codons. This barrier to functional gene transfer during evolution is
greater than any other.
Hydrophobicity hypothesis (HH) Hydrophobicity (measured as “mesohydrophobicity”) of a protein
quantitatively impedes its import into the mitochondrial matrix via TIM23,
which must unfold its cargo fully so as to translocate it without collapsing the
mitochondrial membrane potential. HH does not apply to chloroplasts,
because they lack a membrane potential and so can import folded proteins.
Co-location for redox regulation
(CORR)
Proximity of genes to their products’ site of action facilitates rapid and
microenvironment-specific response of gene expression to metabolic
demands. This is especially advantageous for organellar electron transport
processes and constitutes a barrier to functional transfer (of either cpDNA or
mtDNA genes).

20. Shape of mitochondrial and chloroplast DNA
• In 1960-1990 , it was believed that mtDNA and cpDNA are
circular in shape.
• In 2000-2004 , the methods of extraction, purification and
separation (coupled with pulse field gel electrophoresis) of mtDNA
and cpDNA greatly improved. It was demonstrated that a large
fraction of in-vivo mtDNA and cpDNA in many eukaryotes are
essentially linear in shape, with only small fraction occurring as
circular DNA.
• Linear mtDNA has been reported in some algae (Chlamydomonas),
fungi (yeast), plant (Chenapodium), protozoan (Paramecium).
• Similarly, linear cpDNA was reported in Maize.

21. Size of Mitochondrial and Chloroplast DNA
• The size of mitochondrial DNA differs
greatly among different organisms
ranging from 6kb in plasmodium to
2500kb in Cucumis melo.
• In humans the two strands of ds
mtDNA are distinguished as heavy
strand (H-strand) and light strand (L-
strand). Since they differ in density.
• Chloroplast genome are less variable in
size ranging from 120kb in pea to
204kb in Chlamydomonas.

23. Number of genome per organelle and per cell
• Common belief is that both bacterial cells and chloroplast/ mitochondria contain
their genetic material in the form of single genome circular DNA molecule.
• It is shown that a rapidly dividing E.coli cell on an average contains 11 genome
equivalent of DNA per cell.
• In the same way mitochondria and chloroplast have multi-genomic chromosomes.
• The number of genomes in an individual organism varies is dependent on number
of organelles (chloroplast and mitochondria) per cell.
1. Human cell contains 800 mitochondria and each mitochondria contains 10
identical genomes, so each cell contains 8000 mitochondrial genomes.
2. Yeast contains few mitochondria per cell and each mitochondria contains 100
genomes so each cell contains 5000 mitochondrial genomes.
3. In plants the number of chloroplast genome per cell is 5000.

24. Replication of mitochondrial and chloroplast DNA
• The rolling circle model involving initial formation of D-loop
followed by formation of intermediate θ shaped structures and
finally rolling circles.

25. • Rolling circle model was
recently questioned and
recombination dependent
replication (RDR) has been
proposed for both mtDNA
and cpDNA.

26. GENETIC CONTENT OF
MITOCHONDRIAL GENOME

27. About mtDNA:
• mtDNA constitute 1% of total cellular DNA.
• Multiple(2-10copies) identical circular DNA / linear DNA , with no histones.
• Genes with no introns in humans , few introns in plants.
• Mitochondrial genome exhibit greater variability in gene content ranging
from 5-92.
• Mutation rate is very high.
• Often AT rich genomes.
• Has 2 strand – H (heavy ) and L (light ) strand
• 95 % of mitochondrial proteins are coded by nuclear genes.
• Cytoplasmic male sterility is determined by mt DNA

28. Compared to the mtDNA of yeast, the mtDNA of humans is smaller, contains
significantly less non-coding regions, and does not contain introns

29. Genome Type Kingdom Introns Size Shape Description
1 Animal No 11–28 kbp Circular Single molecule
2 Fungi, Plant, Protista Yes 19–1000 kbp Circular Single molecule
3 Fungi, Plant, Protista No 20–1000 kbp Circular
Large molecule and
small plasmid like
structures
4 Protista No 1–200 kbp Circular
Heterogeneous
group of molecules
5 Fungi, Plant, Protista No 1–200 kbp Linear
Homogeneous group
of molecules
6 Protista No 1–200 kbp Linear
Heterogeneous
group of molecules
There are six main genome types found in mitochondrial genomes, classified by their structure (e.g. circular
versus linear), size, presence of introns or plasmid like structures, and whether the genetic material is a
singular molecule or collection of homogeneous or heterogeneous molecules

30. Mitochondrial genome contains genes for –
1. Non coding RNA – r RNA , t RNA
2. Some Proteins for
 Respiratory chain
 Ribosomal proteins
 proteins involved in transcription and translation.
 code for proteins involved in transport of other proteins into
mitochondria from cytoplasm
3. Number of unidentified reading frame (URFs) , representing
proteins coding genes with unknown product.

31.  13 genes for polypeptide – oxidative phosphorylation
D loop region /control region –contains signals to
control RNA and DNA synthesis
Complex I genes – 7 genes – NADH
Dehydrogenase for electron transfer
Complex III genes – 1 gene –cytochrome b protein
ubiquinol /cytochrome c oxido- reductase.
Complex IV genes -3 genes- cytochrome c oxidase
Complex V genes – 2 genes-ATP synthase
 22 genes – tRNA
 2 genes - rRNA (16s rRNA,12s rRNA)
Human mtDNA
16569bp ----> 37 genes

32. Yeast mtDNA
 Size of genome 85.8 kbp long ---->35
genes
 Mitochondrial DNA is referred to as
dispensable if an organism can survive
without it.
 As a facultative anaerobe, S. cerevisiae
can utilize fermentable carbon sources
as a means of ATP production even if
their mtDNA is massively damaged or
lost altogether. This characteristic of
S. cerevisiae allows researchers to
investigate genomic changes in the
mitochondria that are normally
incompatible with life.

33. Plant mtDNA

34. Algal mtDNA Rice mtDNA

35. Maize mtDNA
• single circular map of 569,630 bp

36. Trypanosomatids are characterized by the
presence of a single mitochondrion, which
contains a limited number of maxicircles,
the equivalent of the mitochondrial DNA of
other eurkaryotes. These maxicircles
contain a limited number of genes involved
in the synthesis of subunits of the
mitochondrial respiratory chain.
It is easily visualized in the light
microscope because of the presence of a
huge kinetoplast DNA network of
maxicircles plus thousands of catenated
minicircles

37. Mitochondrial inheritance
Maternally inherited
• mtDNA is inherited from the mother
• Example : An egg contains on average
2,00,000 mtDNA molecules, whereas a
healthy human sperm has been reported to
contain on average 5 molecules,
degradation of sperm mtDNA in the male
genital tract and in the fertilized egg.

38. Male inheritance:
Paternally inherited mitochondria have additionally been
reported in :
fruit flies, honeybees , periodical cicadas, Plymouth Rock
chickens, mice, sheep, humans.
In exceptional cases, human babies sometimes inherit mtDNA
from both their fathers and their mothers resulting in
mtDNA heteroplasmy (the presence of more than one
mtDNA type in an individual)

39. Significance of mitochondrial genome :
• Useful to trace geographic distribution of genetic variation, for the
investigation of expansions, migrations and other pattern of gene flow.
• Widely applicated in forensic science. It is a powerful implement to
identify human remains.
• Mitochondrial genome is characterized by the high rate of polymorphisms
and mutations. Some of which are increasingly recognized as an important
cause of human pathology such as oxidative phosphorylation (OXPHOS)
disorders, maternally inherited diabetes and deafness (MIDD), Type 2
diabetes mellitus, neurodegenerative disorders, heart failure and cancer.
• Relationship with aging
• In phylogenetic studies.
• Edited plant mitochondrial DNA , which could lead to a more secure food
supply.

40. GENETIC CONTENT OF
CHLOROPLAST GENOME

41. About cpDNA :
• Lack of recombination.
• Stable and conserved.
• It is also known as
the plastome when referring to
genomes of other plastids

42. Codes for about 110-120 genes which includes :
I. Genes for photosynthetic apparatus :
 PS I
 PS II
 Cytochrome b6 –f
 RuBisCO
 ATP synthase
 NADPH dehydrogenase
II . RNA genes and genes for genetic apparatus :
 t RNA
 r RNA
 RNA polymerase
 Ribosomal subunits
III . Open reading frames

43. Presence of 3 regions :
1. 2 inverted repeats (IR) – each 10-24 kb long
carrying ribosomal RNA genes.
2. A short single copy (SSC) sequence – 18-20kb
long.
3. A long single copy (LSC) sequence.

49. Plastid inheritance :
3 modes in Angiosperms:
1. Uniparental maternal – exclusion of male plastid during syngamy.
2. Uniparental biparental –
 plastids preserving their DNA in the generative and sperm cell may also be
transmitted into zygote ,also female plastid are incapable of genetic
transmission (Medicago sativa , Daucus carota)
 DNA replication rate is higher in male plastid than female plastid within the
zygote .
3. Bipaternal plastid inheritance –
 Influenced by control of nuclear genotype
 Ratio of plastid number between male and female cells and plastid
distribution in zygote.

50. Significance of chloroplast genome :
• Chloroplast genome is a rich source for phylogenetic and other comparative data
in plants giving useful information about the relative age and relatedness of
organism possessing them.
• Chloroplast genome would help in rapid, accurate, automated identification of
species using species specific DNA barcode catalogue.
• Chloroplast genome transformation of higher plants has become research hotspot
of plant genetic engineering with several advantages over nuclear genetic
engineering.
• Studies of rearrangement of RuBisCO , structure, transcription, translation and
RNA editing of genes in chloroplast.
• Production of antibodies vaccines like polyβhydroxyl butyrate and bioelastic
protein using chloroplast as bioreactor.
• Generating of transgenic plants with resistance to insect, disease, herbicide,
draught and decreasing the gene flow of transgenic plants.

54. Cytoplasmically inherited traits can be
identified by :
• Inheritance pattern in reciprocal cross is different.
• Absence of segregation at meiosis or presence of type of segregation
which don’t follow Mendelian law.
• Persistent of trait in the progeny after repeated back cross when the
back crossing parent shows an alternative character.
• Mutagens may cause 100% mutations in the plasma genes and thus
they differ in degree when compared to nuclear genes.

55. Promiscuous DNA and Horizontal transfer of genes
 Interactions between the nucleus and
organelles of cytoplasm have a major role in
eukaryotic cellular metabolism.
 The genetic materials of the nucleus,
mitochondrion, and chloroplast are usually
maintained discretely in each organelle
without duplication elsewhere.
 Exceptions are known as promiscuous DNA,
defined as a nucleotide sequence that occurs in
more than one of these three membrane-bound
organelles.

56. 1. Promiscuous DNA (1984) was reported in genomes of mitochondria
and chloroplasts of maize have a 12kb DNA sequence in common.
The most attractive conclusion is that the chloroplast DNA became
incorporated in the mitochondrial genome after gene duplication and
transposition .
2. In Saccharomyces cerevisiae, piece of DNA is common to the
mitochondrial and nuclear genomes. There is good evidence that the
"jumping" occurred in the direction nucleus ^ mitochondrion .
3. The sea urchin, whose sections of mitochondrial DNA, coding for
cytochrome oxidase subunit 1 and 3 , 16 S RNA of ribosomes, are
found in the nuclear genome. This is probably a result of a
chromosomal transposition about 25 million years ago, followed by
chromosomal rearrangements and single nucleotide substitutions .

57. The mechanism of this transfer of the so called promiscuous DNA is not
known but two possibility :
1. Some kind of vector / transposon /transducing phage may be
involved.
2. The two organelles might have undergone fusion at some
stage.

58. Mitochondrial and chloroplast
Genome and Proteome databases.
• The study has been conducted since 1990 until now.
• These details are now available in different databases available at the internet.
• The University of Montreal, Canada maintains the GOBASE database
(http://gobase.bcm.umontreal.ca/) contains information about mitochondrial and
chloroplast genome.
• In June 2010 GOBASE released information which contained
>109 mitochondrial sequence (includes 3,00,000 proteins)
>35 chloroplast sequences (includes 1,20,000 proteins)
• The sequences available in GOBASE are largely derived from GenBank
database.
• However, GOBASE was closed in 2010 due to lack of funds.

59. • A separate database for chloroplast genome is ChloroplastDB
(http://chloroplastcbio.psu.edul) was launched in 2005.
• This database contains information about all fully sequenced plastid
genomes containing proteins, DNA and RNA sequences, gene
locations and RNA editing sites.
• The information about mitochondria and chloroplast genome is also
available at NCBI site as GenBank records.
• On this site list is available for mitochondrial (6000) and plastid
(>200) genome (2014).

60. • Majority of genes encoding proteins for mitochondria and chloroplast
are actually found in the cell nucleus, therefore the information has
been collected for both the following classes of protein :
1. The proteins coded by the organellar genome themselves.
2. The proteins that are coded in the nucleus, and later transported to
organells.
• To facilitate this activity for mitochondrial proteins MITOP database
was created in 1998 at University of Munich in Germany.
• In 2004 MitoP2 database was launched as an extension of MITOP.
• MitoProteome is another database giving information on
mitochondrial genomes.

61. Mitochondrial sequence databases
• AmtDB: a database of ancient human mitochondrial genomes.
• InterMitoBase: an annotated database and analysis platform of protein-protein interactions for
human mitochondria. (apparently last updated in 2010, but still available)
• MitoBreak: the mitochondrial DNA breakpoints database.
• MitoFish and MitoAnnotator: a mitochondrial genome database of fish.
• Mitome: a database for comparative mitochondrial genomics in metazoan animals (no longer
available)
• MitoRes: a resource of nuclear-encoded mitochondrial genes and their products in
metazoa(apparently no longer being updated)
• MitoSatPlant: Mitochondrial microsatellites database of viridiplantae.
• MitoZoa 2.0: a database for comparative and evolutionary analyses of mitochondrial genomes in
Metazoa. (no longer available)
Mitochondrial mutation databases
• Several specialized databases exist that report polymorphisms and mutations in the human
mitochondrial DNA, together with the assessment of their pathogenicity.
• MitImpact: A collection of pre-computed pathogenicity predictions for all nucleotide changes that
cause non-synonymous substitutions in human mitochondrial protein coding genes .
• MITOMAP: A compendium of polymorphisms and mutations in human mitochondrial DNA

62. Organellar DNA Nuclear DNA
1. Found in chloroplast and
mitochondria.
1. Found in chromosomes.
2. Usually circular ,some it is linear. 2. Linear in eukaryotes and circular
in prokaryotes.
3. Synthesis continues throughout
cell cycle.
3. Synthesis occurs only during
interphase.
Difference Between Organellar DNA and Nuclear DNA

63. Bibliography
• Books : Cell and molecular biology – P.K. Gupta
• Websites :
https://en.wikipedia.org/wiki/Mitochondrial_DNA
https://en.wikipedia.org/wiki/Chloroplast_DNA
https://www.slideserve.com/marcel/sizes-of-plastid-cpdna
https://www.slideshare.net/sandeshGM/organellar-genome
• Videos related to mitochondrial DNA and chloroplast DNA.