Download Chapter 21 Extranuclear genes

Chapter 21
Extranuclear genes
A variegated mosaic of Euonymus fortunei.
Green(normal) and albino tissue
caused by mixture of two chloroplast DNA types
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
Components of energy-producing systems
• Organelle genes
Organelle translation component
•
Most organelle-encoded polypeptides
Nucleus-encoded polypeptides
Active proteins
In organelle
Eukaryotic organism DNA
Nuclear chromosome
Mitochondria chromosome
(mtDNA)
Chloroplast chromosome
(cpDNA)
Genes in
Mitochondria : Oxidative phosphorylation
ATP
Chloroplast : Photosynthesis
Content : small
Human nuclear chromosome : 3,000,000 Kb (100,000 genes)
Human mtDNA : 17Kb (37 genes)
Structure of a typical animal cell
Structure of a typical animal cell
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
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
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
Structure of organelle chromosomes
How many copies?
Nuclear chromosome : 1 copy / cell (haploid)
2 copy / cell (diploid)
Organelle chromosome : x00~x,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
Structure of organelle chromosomes
How many copies?
ex.) Mitochondria : haploid yeast
Human
1 ~ 45 mitochondria / cell
2 ~ 10 mtDNA mol. / mitochondria
10 ~ 30 nucleoids / mitochondria
4 ~ 5 mtDNA molecules / nucleoid
MAX : 45 x 30 x 5 = 6750
Nucleus
Mitochondria
Nucleoid
Fluorescent staining of a cell of Euglena gracilis.
Structure of organelle chromosomes
Mitochondrial genomes
Maps of yeast and human mtDNAs
Green : exons and uninterrupted genes
Red : tRNA genes
Yellow : URFs (unassigned reading fames)
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.
Red : Nuclear DNA-encoded subunits
Purple : mtDNA-encoded subunits
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
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
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
>> much less spacer DNA
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
Counterclickwisely transcribe
Clockwisely transcribe
Small single copy region
Inverted repeat region
Large single copy region
Organelle mutations
mtDNA : base-pair-substitution rate
typical mutant phenotype
x10 compare to nuclear DNA
: energy deficiency & slow growth or sickly appearance
cf.) antibiotics resistance : by mutation in rRNA or ribosomal
protein-encoding gene (streptomycin, erythoromycin)
Neurospora : Obligate aerobe - can’t 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
Organelle mutations
Deleted DNA region of petite is amplified through
tandem duplication to provide the normal size
Human : Several diseases caused by mtDNA mutation (mitochondrial cytopathies)
>> effected organ - high energy demanding organ (muscle, nerves)
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
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
-> phenotypically similar to cpDNA mutation, but they are
inherited in a strict Mendelian manner, whereas cpDNA
mutation inherited in the non-Mendelian manner
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
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
Petite
mutation
Wild-type
cells
Fused
Petite
Phenotype
Petite-causing mitochondria (increased)
Neurospora
(deletion of mt genes while duplication
of replication origins, etc..)
: 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%
heart
40%
skeletal muscle
50%
cerebellum of brain 14%
liver
14%
kidney
40%
frontal lobe of brain 44%
Threshold effect!
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
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.
Inheritance of organelle genes and mutations
Maternal inheritance
Maternal inheritance of poky Neurospora
Mating
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 - 1:1 Mendelian ratio
Poky female x wild-type male
All progeny poky
Wild-type female x poky male
All progeny wild type
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-o’clock plant.
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.
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 matall progeny sm-r
sm-s mat+ x sm-r matall progeny sm-s
Chlamydomonas reinharditi.
Pair of flagella and the
large single chloroplasts
Uniparental inheritance
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 baker’s yeast (Saccharomyces cerevisiae).
Next slide figure
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 
>> Mendelian pattern 1:1
Drug resistancy >>Uniparental 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)
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.)
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!
Mitochondria and aging
Wear-and-tear theory
: one of the theories of the mechanism of aging
damage
damage
repair machines
Do not fully repair
damage
Repair
Die
Machine can’t function
Repair
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
mtDNA
B ages
A ages
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
Recombination of extranuclear DNA
Cytoplasmic male sterility
Mitochondria and aging