characteristic features of extra nuclear genome

1. EXTRA NUCLEAR
GENOME
P HARITHA
DL BOTANY
TTWRDC M
SRD

2. Introduction:
 In eukaryotes, the nucleus is the primary
organelle for storing the genetic
information of the cell.
 Here, this information is transcribed to
form the RNA, which is modified and is
later transported to the cytoplasm.
 The m RNA is translated in the cytoplasm
and the proteins are formed.
 These proteins may take part either in
catabolic or anabolic processes of the cell.
 However, certain organelles of the cell
other than nucleus also contain their own
genome.

3.  These organelles are the mitochondria and
chloroplasts present in the cytoplasm.
 They play an important role in cytoplasmic
inheritance of characters.
 The genes located in the chromosomes of
the nucleus are called nuclear genes or,
more commonly, simply as genes.
 All other types of genes found outside the
nucleus are referred to as the extra
nuclear genome.

4. Chloroplast DNA (Cp DNA):
 Chloroplasts contain naked circular DNA
molecules of about 140 kb in higher plants and
<200 kb in lower eukaryotes.
 The chloroplast genome has been completely
sequenced in liverworts and tobacco.
 The size of Cp DNA is between 120-155 kb.
 Chloroplasts contain DNA dependent RNA
polymerase.
 A single DNA molecule constitutes the
chloroplast genome in most species.
 But in some brown algae, a chloroplast may
consists of more than one copy of DNA
molecule.

6.  Each chloroplast has 10-60, in some plants
upto 100, copies of its genome.
 They are arranged in 5-6 specific regions
within a chloroplast.
 Chloroplasts of all higher plants and lower
eukaryotes contain DNA.
 Chloroplast genomes are relatively small in
size ranging from 120 to 160 kb in most plant
species.
 The circular Cp DNA molecule exhibits a
characteristic feature.
 It has a 10 to 24 kb long 1 sequence that is
present in two identical copies per genome as
an inverted repeat(IR).

8.  Thus the genes located in the repeat are
present in two copies per genome; they
include r RNA genes.
 The repeat divide the genome into a short
single copy(SSC) and a large single
copy(LSC) regions.
 Chloroplast DNA code for about 120
genes, out of which 20 genes have introns
in them.
 Majority of the genes are present in LSC
region.

10.  Among the identified genes, 45 genes code
for RNA, 27 code for proteins involved in
gene expression, 18 encode for proteins of
thylakoid membrane and 10 produce
proteins concerned with redox reactions.
 A substantial amount of DNA is junk, not
coding for any genes.
 Chloroplast DNA does not associate with
histone protein and thus exist within the
organelle as a naked fibre.
 Chloroplast characteristics are controlled by
both Cp DNA as well as nuclear DNA.

11.  The Cp DNA encodes all the RNA species and
some proteins involved in chloroplast function.
 The genes present in Cp DNA are transcribed
and translated within the chloroplasts.
 The machinery for replication of Cp DNA is
provided by nuclear genes, while plastid rRNA
is produced by Cp DNA itself.
 Most other components of plastid structure and
function are controlled by Cp DNA and nuclear
DNA.

12.  Ribosomes of chloroplast shows
sedimentation coefficient of 70S Svedberg
units i.e., 70S.
 The 70S ribosomes are made up of 23S
and 16S rRNAs.
 Their RNAs are synthesized from
chloroplast DNA template.
 Presence of 70S ribosome in chloroplast
and resemblance to prokaryotic ribosome
strengthen the hypothesis of its
prokaryotic origin.

13. Mitochondrial DNA( mt DNA):
 Mitochondria are filamentous or granular
shaped cell organelles present in the
cytoplasm of eukaryotic cells.
 Mitochondria act as power house in order
to meet unprecedented demand for ATP
energy.
 They are freely dispersed in cytosol
carrying substantial amount of ATP energy.
 Within the mitochondrial matrix small
ribosomes and circular DNA molecules are
present.

15.  The mitochondrial DNA is depicted as mt
DNA.
 Mitochondrial DNA was discovered in 1960s
and the advent of r DNA technology made it
possible to analyse mt DNA in great detail.
 The complete nucleotide sequences of mt
DNA of many species have now been
determined.
 The mt DNA is a circular molecule and
varies from n 16.5 kb (mammals) to100 kb
or more(higher plants) in length.

16.  The G+C content of mt DNA shows
considerable variation from one species to
another, i.e., 18% in yeast and 47% in
higher plants.
 The mt DNA encodes all the RNA copies
and some proteins needed for
mitochondrial function.
 The genes are transcribed and translated
within the mitochondria.
 But several proteins are contributed by
nuclear DNA.

17. Mitochondrial DNA under electron
microscope

18. A) Yeast mitochondrial Genome:
 Mitochondrial genome of yeast and
several other fungi is about 78 kb long.
 It has both split( oxi 3, ATPase 9 and
21S r RNA) and uninterrupted genes.
 Mt DNA encodes for two ribosomal RNA
molecules(15S and 21S r RNA),
complete range of t RNA molecules and
mRNA for the synthesis of nine proteins.
 The genes for two rRNA are situated on
separate portions of the genome.

20.  Mitochondria alone cannot carry out all its
genetic activity and other functions.
 Most of its key functions are controlled by
nuclear DNA.
 The polymerase for the synthesis of
mitochondria DNA and RNA and nearly all
the mitochondrial ribosomal proteins are
encoded by nuclear genes and synthesized
in cytoplasmic region.
 Genes for the synthesis of enzymes for
kreb’s cycle and electron transport chain
are encoded by nuclear genes.

21.  All these proteins are made in the
cytoplasm and are transported later.
 The mt DNA encodes for 24 t RNA’s.
 These genes have organised in operon
fashion.
 The genetic code of mitochondria differs
from that of the nucleus in several
aspects.
 For example, UGA specifies tryptophan in
mitochondria in mitochondria in place of
chain termination(nuclear code).
 Similarly, AUA codes for methionine rather
than isoleucine.

22. B) Mammalian Mitochondrial Genome:
 Mitochondrial genome of human, rat and
mouse have been completely sequenced.
 They are similar in size(16.5 kb).
 This small mitochondrial genome codes for
limited number of proteins.
 Several mitochondrial proteins are
encoded by nuclear genome and are
synthesized on cytoplasmic ribosomes.
 Human mitochondrial DNA has several
sets of genes like 22 t RNA genes, 2r RNA
genes and 13 protein coding regions.

24.  Expression of some of these genes are in
clockwise and some are in counter clock
wise directions.
 The proteins which are concerned with
respiration are cytochrome b, three
subunits of cytochrome oxidase, one of
the subunit of ATP ase and seven subunits
of NADH dehydrogenase.
 The organization of mammalian
mitochondrial genome is highly compact.

25.  Introns are not found and some genes are
overlapped.
 Some of the genes do not even have the
complete stop codon at the 5’ end of the
coding region.
 The reading frames of these genes end
with either U or UA so that the ochree
codon is generated when a short poly A
tail is added to the 3’ end.
 Mitochondrial DNA can be separated into
two strands based on their different
densities.

26.  One of the strand is referred as heavy
strand(H strand) and other is light
strand(L strand).
 Most of the genes are distributed on
heavy strand, whereas L strand seems to
contain genes for a few t RNA and single
m RNA molecule.
 In clockwise transcription, the H strand is
transcribed, while the L strand is
transcribed in anti clockwise direction.

27.  The amino acid composition of
mitochondrial protein are hydrophobic.
 The r RNAs in mammalian mitochondrial
DNA are smaller than those made in yeast
having 955 and 1550 nucleotides
responsible for 12S and 16S rRNA
respectively.
 Mammalian mitochondrial DNA exhibit
some difference in their genetic code.
 AUG codes for methionine and can be
used as an initiation codon.

28.  However, it does not employ N-formyl
methionine for initiation unlike fungal
mitochondria.
 Apart from this, codon AUU codes for
isoleucine.
 It is also used as initiation codon.
 The triplet codon UGA is used for
tryptophan rather than for termination.
 In mammalian mitochondrial DNA, AGA and
AGG act as termination codons rather than
codons for arginine.

29. Features of organelle genomes:
 They are circular molecules of DNA. In few
cases, mt DNA is linear.
 They are present in multiple copies in
each organelle. In higher plants, Cp DNA
is present in 20-40 copies per chloroplast.
Yeast cells have 4 genomes per
mitochondrion.
 They encode all the RNA species and some
of the proteins required for organelle
function.
 Both Cp and mt DNA are transcribed and
translated within the organelle.

30.  Plant chloroplast contains circular DNA of
190 kb. Chloroplast genes contains introns
and splicing mechanism. Where as
mitochondrial DNA is smaller in size(16.5
kb) and shows no introns.
 Based on DNA density, two chains are
present in mammalian mt DNA. They are
heavy chain(H) and light chain(L). Most of
the genes are distributed on heavy chain.

31. Plasmids:
 A plasmid is a circular DNA molecule,
other than the bacterial chromosome, that
is capable of independent replication and
transmission.
 Most, but not all, plasmids are
dispensible as they are not essential for
the survival of bacterial cells,except under
certain environmental conditions.
 Some plasmids are capable of becoming
integrated into the bacterial chromosme,
they are called episomes.

32.  Plasmids vary in size from those having
only three genes to those containing
several hundred genes.
 Bacterial cells may harbour zero to
several(upto 11) different plasmids.
Copy number:
 Each plasmid is maintained in the
bacterial cell at a characteristic copy
number, mainly due to its replication
control system.
 In this respect, plasmids are of the
following two types.

33. Single copy plasmids:
 They are maintained in one copy per cell.
 Their replication is the same as that of the
bacterial host chromosome.
 So they replicate and segregate along with
the bacterial chromosome.
 Such a replication control is called
stringent control.
Multi copy plasmids:
 They exist in several, but a characteristic
number of copies(10-20) per cell.

34.  Their replication control is independent of
replication of host cell chromosome.
 This permits more than one replication to
occur.
 This type of replication control is often
called relaxed control.
Compatibility Group:
 The regulation of plasmid copy number
gives rise to plasmid compatibility group.
 A plasmid compatibility group includes all
those plasmids whose members cannot be
maintained together in the same host cell.

35.  Therefore, those plasmids that can be
maintained together in the same cell
belong to different compatibility groups.
 It is believed that plasmids belonging to a
single compatibility group share the same
mechanism of replication control.
Types of plasmids:
 There are several types of bacterial
plasmids, of which the following three
have been extensively studied: (1) F
plasmids, (2) R plasmids and (3) Col
plasmids.

36. F plasmids:
 These plasmids carry genes for the
development of F pili(sex pili) and are
responsible for conjugation.
R plasmids:
 These were earlier called resistance
transfer factors.
 They carry genes for resistance to
antibiotics or other antibacterial drugs.
Col plasmids:
 These were earlier called colicogenic
factors and code for colicins, the proteins
that kill sensitive E.coli cells.

37.  They also carry genes that provide
immunity to the particular colicins.
 Other types of plasmids are degenerative
plasmids(they enable the host bacterium
to metabolize unusual molecules like
toluene and salicylic acid e.g. Tol plasmid
of Plasmodium putida) and virulene
plasmids(these confer pathogenicity on
their host bacteria. E.g. Ti plasmid of
Agrobacterium tumefaciens).
 Plasmids may be either(1) Conjugative or
(2) Non conjugative.

38.  Conjugative or transmissible plasmids mediate
the transfer of DNA through conjugation.
 They carry ‘tra’ (for transfer) genes that
promote conjugation.
 E.g. F and F’ plasmids , many R plasmids and
some Col plasmids.
 These plasmids rapidly spread among the cells
of a bacterial population as does the F
plasmids.
 The rapid spread of conjugative R plasmids is
responsible for many pathogenic bacteria
becoming resistant to many widely used
antibiotics, such as pencillin, tetracycline,
streptomycin, kanamycin, etc.

39.  In contrast, non-conjugative or non-
transmissible plasmids do not mediate
DNA transfer through conjugation.
 These plasmids can only be transferred
with the assistance of conjugative
plasmids by ‘accident’. E.g. many R and
most Col plasmids.
 Plasmids and episomes contain insertion
sequences(IS), which are also present in
bacterial chromosomes.
 IS sequences are transposable in that
they can move about within and between
chromosomes.

40.  Thus, they mediate genetic recombination
between otherwise non homologus genetic
elements within which they (IS elements)
are located.
 Such transpositions generate R plasmids
having resistance genes for several
antibiotics.
Structure of typical plasmid vectors:
pBR 322:
 The name pBR denotes the following: p
signifies plasmid, while B and R are derived
from Boliver Rodriguez, the scientist who
developed p BR 322.

42.  It is the most popular and most widely
used plasmid of 4362 bp.
 Its entire base sequence is known.
 It has the replication module of E.coli
plasmid col E1.
 This module has been incorporated in
many other plasmid vectors, since it
permits plasmid replication even when
chromosome replication and cell division
are inhibited by amino acid starvation or
chloramphemicol.
 Under such conditions, each cell
accumulates several thousand copies of
the plasmid.

43.  This plasmid has two selectable marker
genes, tetracycline (tetr) and
ampicillin(ampr) resistance genes, and a
single unique recognition site for 12
different restriction enzymes.
 The recognition site for two of the
restriction enzymes(Pst1 and Pvu1) is
located within the ampr gene and for
Bass, Hl, Sal I etc. are located within tetr
gene.
 The presence of restriction sites within the
markers tetr and ampr permits an easy
selection for cells transformed with the
recombinant p BR 322.

44.  The insertion of the DNA fragment into the
plasmid using restriction enzyme Pst I or
Pvu I, places the DNA insert within the
gene ampr.
 This makes the ampr gene non functional.
 Bacterial cells containing such as
recombinant pBR 322 will be unable to
grow in the presence of ampicillin, but they
will grow on medium containing
tetracycline.
 Similarly, when restriction enzyme Bass HI
or Sal I is used, the DNA insert is placed
within the gene tetr making it non
functional.

45.  Bacterial cells possessing such a
recombination p BR 322 will, therefore grow
on ampicillin, but not on tetracycline.
 This feature allows an easy selection of a
single bacterial cell having recombinant pBR
322 among 10 power 8 other types of cells.
Ti plasmid:
 These plasmids are found in tumour cells
produced by Agrobacterium tumefaciens in
the roots of plants.
 These plasmids act as natural gene vectors,
which can transfer, incorporate and express
the genetic information in higher plants.

47. Role of plasmids:
 The naturally occuring plasmids(cryptic
plasmids) are symbiotic to the host and
provide some useful characters in their host.
 Some of these characters are, antibiotic
resistance capability to degrade complex
organic compounds, and thus making the
host more suited to survive under adverse
condition.
 Several plasmids serve as important tools in
genetics and biochemistry. The propagative
plasmids are commonly used as vectors for
cloning plasmids. e.g. Ti plasmid, PUC, PBR
322.

48.  Plasmids make large number of copies of
a particular gene by which several desired
products can be generated. E.g. Insulin,
antibiotics.

49. THANK YOU