Chapter 21 Extranuclear genes

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Chapter 21 Extranuclear genes
A variegated mosaic of Euonymus
fortunei. Green(normal) and albino tissue caused
by mixture of two chloroplast DNA types
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Key concepts

• Chloroplast and mitochondria
• Their own unique chromosome of genes
• Organelle DNA and their phenotype maternally
  inherited
• Sorting-out process
• Dihybrid organelle mixture ? Recombination can
  be detected
• Organelle genes
• Most organelle-encoded polypeptides

Components of energy-producing systems Organelle
translation component
Active proteins In organelle
Nucleus-encoded polypeptides
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Nuclear chromosome Mitochondria chromosome
(mtDNA) Chloroplast chromosome (cpDNA)
Eukaryotic organism DNA
Genes in Mitochondria Oxidative
phosphorylation Chloroplast Photosynthesis
ATP
Content small Human nuclear chromosome
3,000,000 Kb (100,000 genes) Human mtDNA 17Kb
(37 genes)
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Structure of a typical animal cell
Structure of a typical animal cell
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Origin of extranuclear genes

• Generally believed

Endosymbionts
Lost
Integration
Most modern eukaryotic cells
Fully dependent on the organelle genes for their
normal function cf.) The yeast, Saccharomyces
cerevisiae
Without mitochondria obtain energy from
fermentation
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Structure of organelle chromosomes
Protocol for studying
Organelle DNA fraction preparation
Standard Recombination DNA technology
Several organelle chromosomes fully
sequenced Functions of organelle gene
Mutation analysis homology searching
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Structure of organelle chromosomes
Overall organization

• Difference with nuclear chromosomes

Circular form by restriction
mapping electron microscope
Not in the highly condensed form Not in a
euchromatic state
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Structure of organelle chromosomes
How many copies?
Nuclear chromosome 1 copy / cell
(haploid)
2 copy / cell (diploid) Organelle
chromosome x00x,000 copy/ cell
regulation of
copy number is relatively loose ex.)
Chloroplast Leaf cells of the garden beet
40 chloroplast /
cell 4 8
nucleoids / chloroplast
(nucleoid Specific heavily DNA
area in chloroplast)
4 18 cpDNA molecules / nucleoid
MAX 40 x 8 x 18 5760
copy of cpDNA / cell
Chlamydomonas
1 chloroplast / cell 500
1500 cpDNA molecules
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Structure of organelle chromosomes
How many copies?
ex.) Mitochondria haploid yeast
1 45 mitochondria / cell
10 30 nucleoids / mitochondria
4 5 mtDNA molecules / nucleoid
MAX 45 x 30 x 5 6750
Human 2 10 mtDNA mol. / mitochondria
Nucleus
Mitochondria
Nucleoid
Fluorescent staining of a cell of Euglena
gracilis.
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Structure of organelle chromosomes
Mitochondrial genomes
Maps of yeast and human mtDNAs
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Structure of organelle chromosomes
Mitochondrial genomes
Function
Some of the proteins oxidative
phosphorylation tRNA, rRNAs, some proteins
mitochondrial protein synthesis
(some
genes are still encoded in nucleus)
mRNA is translated outside the mitochondria on
cytosolic ribosomes
Synthesized proteins are transported into the
mitochondria
Complete system is assemble in mitochondrial
inner membrane
The mitochondrial respiratory chain.
Purple mtDNA-encoded subunits
Red Nuclear DNA-encoded subunits
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Structure of organelle chromosomes
Mitochondrial genomes
tRNA
25 yeast, 22 human tRNA in mitochondria at
least 32 tRNA for nucleus-derived mRNA
tranlation
? tRNA of mitochondria more wobble
And codon assignments is different with nuclear
codon with different species
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Structure of organelle chromosomes
Mitochondrial genomes
Differ with nuclear code
The genetic code of the human mitochondria. 22
tRNA types by the 22 boxes that do not contain
stop codons
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Structure of organelle chromosomes
Mitochondrial genomes
Yeast
The intron in several mitochondrial gene
Ex.) Subunit I of cytochrome oxidase - 9 introns
nuclear gene - rare intron The
existence of unassigned reading frames (URFs)
within the yeast intron
URF - sequences that have correct initiation
codons are uninterrupted by stop codons some
URF - important in the splicing out of the
introns themselves at the RNA level ?
Specifying proteins
Human
Much smaller more compact than yeast mtDNA gtgt
much less spacer DNA
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Structure of organelle chromosomes
Chloroplast genomes
cpDNA 120 200Kb in different species ex.)
Liverwort Marchantia 136 genes
4 kinds of rRNA 31 kinds of tRNA 90 proteins (20
photosynthesis electron-transport
functions) half the chloroplast genome relate
with translational function
Large inverted repeats of virtually all species
of plants (the sequences of the repeats are
same!) Like mtDNA, cpDNA cooperates with nuclear
DNA to provide subunits for functional proteins
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Counterclickwisely transcribe
Clockwisely transcribe
Small single copy region
Inverted repeat region
Large single copy region
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Organelle mutations
mtDNA base-pair-substitution rate x10
compare to nuclear DNA
typical mutant phenotype
energy deficiency slow growth or sickly
appearance cf.)
antibiotics resistance by mutation in rRNA or
ribosomal
protein-encoding gene (streptomycin,
erythoromycin)
Neurospora Obligate aerobe - cant survive
without functional mt Yeast can obtain ATP by
fermentation - drastically deleted genotypes can
survive Small colonies caused
by
petites - deletion of part or even all of the
mtDNA mit mutation - point mutations in some
electron-transport proteins
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Organelle mutations
Human Several diseases caused by mtDNA
mutation (mitochondrial cytopathies) gtgt effected
organ - high energy demanding organ (muscle,
nerves)
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Organelle mutations
Map of human mtDNA showing loci of mutations
leading to cytopathies. Single letter are
one-letter abbreviations for amino acids. ND,
NADH dehydrogenase COX, cytochrome oxidase 12S
and 16S, ribosomal RNAs
Point mutations or Large deletions
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Organelle mutations
Model for producing a deletion by crossing-over
in a direct repeat
Mutations in nuclear genes can result in white
leaves Cannot photosythesize -gt phenotypically
similar to cpDNA mutation, but they are inherited
in a strict Mendelian manner, whereas cpDNA
mutation inherited in the non-Mendelian manner
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Inheritance of organelle genes and mutations
Inheritance
Expression many copies Cytoplasmic segregation
division of heteroplasmon (cytohet)
Maternal inheritance Between heterogametes
The genetic fate of an organelle DNA mutation
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Inheritance of organelle genes and mutations
Expression of organelle mutations
Possibility

• Suppressive organelle mutations (not mean as
  suppressor)
• outreplicate the wild-type organelle
  genomes within a cell

Yeast
Neurospora abn (abnormal) mutation (rapid
express mut mt gene even in 1/10,000 ratio of
mutants to wt in heteroplasmic mycelium)
Human
in post-mitotic cells different parts of the
body contain different proportions
smooth muscle 4
liver 14
heart 40
kidney 40
skeletal muscle 50
frontal lobe of brain 44
cerebellum of brain 14
Threshold effect!
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Inheritance of organelle genes and mutations
Expression of organelle mutations

• Random drift the frequency of mtDNA types can
  rise and fall
• entirely on the
  basis of chance

• certain type of mutation of mitochondria
• recognize potential energy deficiency
• start replication faster

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Inheritance of organelle genes and mutations
Cytoplasmic segregation
the production of mutant and wild-type
descendant cells of a heteroplasmon
Heterokaryon test
Fused
Not to mix between sg and sg-
The heterokaryon test is used to detect
extranuclear inheritance in filamentous fungi.
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Inheritance of organelle genes and mutations
Maternal inheritance
Maternal inheritance of poky Neurospora
Poky determinants
Reciprocal crosses of poky (slow growing) and
normal Neurospora. Female the parent
contributing most of the cytoplasm of the progeny
cells Nuclear locus - 11 Mendelian ratio
Poky female x wild-type male Wild-type female x
poky male
All progeny poky All progeny wild type
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Inheritance of organelle genes and mutations
Maternal inheritance
Maternal inheritance of chloroplast pigments in
plants
1909 Carol Correns Varigated expression of
chloroplast in leaf Normal flowers ? mating
maternal inheritance
Leaf variegation in Mirabilis japalpa, the
four-oclock plant.
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Inheritance of organelle genes and mutations
Maternal inheritance
Maternal inheritance of chloroplast pigments in
plants
A model explaining the results of the Table 21-1.
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Inheritance of organelle genes and mutations
Maternal inheritance
Uniparental inheritance in Chlamydomonas
reinharditi.
Streptomycin-sensitive mutant (sm-s) Mating type
gene (mat)
sm-r mat x sm-s mat-
all progeny sm-r sm-s mat x sm-r mat-
all progeny sm-s
Uniparental inheritance
Chlamydomonas reinharditi. Pair of flagella and
the large single chloroplasts
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Inheritance of organelle genes and mutations
Uniparental inheritance of mitochondrial
mutations in budding yeast
mtDNA inheritance not dependent on mating type
? uniparental inheritance
Ex) uniparental inheritance in neutral petites
mtDNA petite x wild-type ? all wild-type progeny
N.B. (suppressive petites) suppressive petites x
wild-type ? petite progeny in proportions that
correlate with the degree of suppessiveness
Thus, petites are atypical example of
uniparentality In mtDNA inheritance ?
Drug-resistant mutation and mit point mutations
clearly show uniparental inheritance pattern
The life cycle of bakers yeast (Saccharomyces
cerevisiae).
Next slide figure
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Inheritance of organelle genes and mutations
Uniparental inheritance of mitochondrial
mutations in budding yeast
The special inheritance pattern certain
drug-resistant phenotypes in yeast. eryR and eryS
erythromycin resistance, sensitivity,
respectively
Nuclear genes, mating-type alleles a and ? gtgt
Mendelian pattern 11
Drug resistancy gtgtUniparental inheritance
Maternal inheritance of human cytopathies.
Usually mtDNA deletion are de novo in origin and
are not inherited maternally But, various point
mutations inherited maternally (ex., MERRF
mutation)
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Recombination of extranuclear DNA
Recombination in mitochondrially dihybrid
heteroplasmons mitochondrial fusion
crossing-over-like process
Recombination
Demonstrate this process by drug resistance
Drug-registance alleles (eryR erythromycin re.
spiR spiramycin re.)
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Cytoplasmic male sterility
Male sterility in plant - cytoplasmical base
- maternally inherited
Double-cross hybrids ? Prevent
self-pollination
The use of cytoplasmic male sterility to
facilitate the production of hybrid corn.
Larger and more vigorous!
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Mitochondria and aging
Wear-and-tear theory one of the
theories of the mechanism of aging
Die
damage
repair machines Do not fully repair
Machine cant function
damage
damage
Aging process
Mitochondria - Reduction in oxidative
phosphorylation
Accumulation of deletion and point mutations
Mitochondria replacment Age dependent
correlation in oxidative phosphorylation
Ex..
A ages
B ages
A ages
mtDNA
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Origin of extranuclear genes Structure of
organelle chromosomes - overall
organization - how many copies? -
mitochondrial genomes - chloroplast
genomes Organelle mutations Inheritance of
organelle genes and mutations - expression
of organelle mutations - cytoplasmic
segregation - maternal inheritance Recombina
tion of extranuclear DNA Cytoplasmic male
sterility Mitochondria and aging