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Organelle Genetics I & II (Lectures 8&9) Flashcards

1
Q

Understanding Organelle DNA heredity: 7

A
  1. *a type of EXTRANUCLEAR inheritance
  2. *NON-MENDELIAN inheritance
  3. *UNIPARENTAL inheritance
    1. *typically MATERNAL
      1. *female contributes BULK OF CYTOPLASM TO PROGENY
      2. *are EXCEPTIONS; PARENTAL, BIPARENTAL

7.*NO SEGREGATION RATIOS AS FOR NUCLEAR GENES

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2
Q

UNDERSTANDING Chloroplast DNA Heredity = 6

A
  1. *VARIEGATED PLANTS
    1. *white regions
      1. *CELLS WITH MUTATION IN A GENE CODING FOR A PROTEIN INVOLVED IN CHLOROPHYLL SYNTHESIS
        4. *gene is in chloroplast genome (cpDNA)
  2. *chloroplast GENES IN FLOWERS ARE SAME as those ON SUPPORTING BRANCH
  3. *e.g., white branch - male and female
    gametes will have cpDNA with
    MUTATION
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3
Q

Chloroplast DNA Heredity PROCESS: 4

A
  1. *STRICT MATERNAL INHERITANCE seen for ZYGOTES WHERE EGG CELL is FROM ‘NON-VARIEGATED’ BRANCH.
    • EGGS CELLS FROM ‘VARIEGATED BRANCH’ may have cpDNA WITH MUTATION or WT cpDNA, OR A MIXTURE (‘CYTOHETS’)
    • ZYGOTES CONTAINING BOTH TYPES OF cpDNA often show ‘CYTOPLASMIC SEGREGATION’ as they DIVIDE
       4. *both WT and mutant cpDNA containing cells and tissues (a variegated plant)
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4
Q

Chloroplast DNA Heredity DIAGRAM

A

SLIDE 6

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5
Q

cytohets (heteroplasmons) ?

A

*cytohets (heteroplasmons)

*cells with a mixture of organelle genomes

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6
Q

Cytoplasmic Segregation IN CYTOHETS = 4

A
  1. *cytohets (heteroplasmons)
    *cells with a mixture of
    organelle genomes
    • FOLLOWING MITOSIS*
      *PROGENY WITH MIXTURE
      * PROGENY WITH ONE OR OTHER —> ‘CYTOPLASMIC SEGREGATION’
  3. *CHANCE EVENTS
  4. *ORGANELLES DO NOT SEGREGATE TO POLES ALONG MITOTIC SPINDLE, ‘STOCHASTIC PARTIONING INSTEAD’

DIAGRAM IN SLIDE 7

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7
Q

Cytoplasmic Inheritance in Humans: 6

A
  1. *number of mutations in mitochondrial genes that can cause disease
    • HUMAN PEDIGREES SHOW PHENOTYPES TRANSMITTED FROM MOTHERS TO SONS AND DAUGHTERS…
  3. *NOT ALL COPIES OF MITOCHONDRIAL DNA (mtDNA) IN A CELL WILL HAVE THE MUTATION.
    • SEVERITY OF DISEASE ASSOCIATED WITH PROPORTION OF MUTATED mtDNA INHERITED.
  5. *2018 (Luo et al. PNAS 115: 13039) –
    EVIDENCE OF BIPARENTAL INHERITANCE of MITOCHONDRIAL DNA in HUMANS
  6. *DEEP SEQUENCING INDICATES biparental inheritanceMORE COMMON THAN ANTICIPATED
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7
Q

Cytoplasmic Inheritance in Humans

‘Myoclonic epilepsy and ragged red fibre
(MERRF) disease’

A
  1. *lack of muscle coordination, deafness, dementia
  2. *“ragged red” - muscle fibre appearance
  3. *single base change leading to mutation in mitochondrial tRNA(Lys)
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8
Q

Cytoplasmic Inheritance in Humans:

‘Leber hereditary optic neuropathy (LHON)’

A
    • SUDDEN BILATERAL BLINDNESS
  2. *4 mutations identified - all lead to disruption of OXIDATIVE PHOSPHORYLATION
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9
Q

Cytoplasmic Inheritance in Humans – Reconstructing Relationships Among Populations……..mtDNA…

WHY IS MITOCHONDRIAL DNA IMPORTANT? = 6

A

mtDNA
1. *good genetic marker for tracing human ancestry

  1. *little or NO RECOMBINATION
  2. *EVOLVES AT FASTER RATE than nuclear DNA (GOOD FOR STUDYING CLOSELY RELATED GROUPS)
  3. *1 change per mitochondrial lineage every 3800 years
  4. *maternally inherited
    • CAN ESTIMATE THE NUMBER OF YEARS SINCE POPULATIONS HAVE BEEN SEPARATED
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10
Q

Cytoplasmic Inheritance in Humans – Reconstructing Relationships Among Populations

mtDNA … ANCESTRAL RELATIONSHIPS?

MOST RECENT ANCESTOR?

A
  1. NUCLEOTIDE DIFFERENCES in mtDNA USED TO CONSTRUCT ANCESTRAL RELATIONSHIPS

—— *3 of 4 major lineages from subsaharan Africans

——*age of most recent common ancestor (MRCA)
~170,000

—— *age of MRCA of lineage joining African & non-African populations ~50,000 years

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11
Q

Division and Segregation of Organelles - Chloroplasts: 12

A
  1. *chloroplasts come from PRE-EXISTING chloroplasts
  2. *requires INTERACTION of PROKARYOTE-DERIVED and EUKARYOTE-DERIVED MACHINERIES
  3. *FtsZ ring
    *FtsZA, FtsZB (filamentous temperature-sensitive) proteins
    *bacterial cell division proteins
    *form a ring inside chloroplast, lining inner membrane surface
  4. *plastid-dividing ring (PDR)
    *nanofilaments (polyglucan)
    *eukaryotic origin
    *form inside and outside
    organelle
  5. *dynamin ring
    *dynamin-related protein
    (eukaryotic membrane REmodeling GTPases)
    *forms ring outside the
    chloroplast
  6. PDR and dynamin rings twist to
    pinch membrane
  7. *chloroplasts interact with cytoskeletal
    components during cytokinesis
  8. *details of segregation not known
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11
Q

Division and Segregation of Organelles - Mitochondria = 6

A
  1. *FtsZ ring
    *forms a ring inside mitochondrion, lining the inner membrane surface
  2. *mitochondrial-dividing (MD) rings
    *form inside and outside organelle
    *nanofilaments (polyglucan)
    *eukaryotic origin
  3. *dynamin ring
    *forms outside organelle
    *eukaryotic origin
  4. *MD and dynamin rings twist to pinch membrane
  5. *mitochondria interact with cytoskeletal components during cytokinesis
    1. *details of segregation not known
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12
Q

Structure of Mitochondrial Genomes SIZES IN VARIOUS ANIMALS

A

Mitochondrial DNA (mtDNA)

*animal; 15 - 18 kb

*yeast; 75 - 90 kb

*plant; 200 - 2500 kb

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13
Q

Structure of Mitochondrial Genomes: 5

A
  1. *higher plant mtDNA exhibits high levels of recombination
  2. *crossing over between large repeat regions
  3. *leads to multiple circular “chromosomes” of different sizes
    • The coding capacity of the genome may be distributed among these subgenomic molecules
    • The number of subgenomic molecules may vary within a mitochondrion
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14
Q

diagram : Structure of Mitochondrial Genomes

A

slide 15

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15
Q

Structure of Mitochondrial Genomes: how many copies? recombination?

A

*multiple copies of mtDNA per organelle, typically multiple organelles per cell

*recombination DOES occur – heteroplasmy

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16
Q

Structure of Mitochondrial Genomes: CODING CAPACITY = 6

A

*coding capacity
*50 – 60 genes

*only four genes common to all known
mitochondrial genomes:
1 *cob cytochrome b
2 *cox1 cytochrome oxidase subunit
3 *rns and rnl rRNAs

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17
Q

RECLINOMONAS

A

ANCESTRAL - GREAT NUMBER OF GENES IN MITOCHONDRIAL GENOME

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18
Q

PLASMODIUM

A

HIGHLY DERVIED - FEWEST GENES

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19
Q

Basic Genetic Mechanisms of Mitochondrial Genomes: 3

A
  1. *genes on both strands
  2. *“machinery” for replication, transcription & translation encoded by mtDNA and nuclear genome
  3. *gene products from both genomes are required for functional organelles
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20
Q

Basic Genetic Mechanisms of Mitochondrial Genomes IMPORTANT DIAGRAM

A

SLIDE 18

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21
Q

Basic Genetic Mechanisms of Mitochondrial Genomes
‘ Replication’ =

A
  1. *mtDNA synthesised throughout cell cycle, NOT COORDINATED with synthesis of nuclear DNA
  2. *which molecules are replicated is RANDOM
  3. *within an organelle some molecules replicated MULTIPLE TIMES, OTHER NOT REPLICATE r
22
Q

Basic Genetic Mechanisms of Mitochondrial Genomes
‘Replication’ IMPORTANT DIAGRAM

23
Q

Basic Genetic Mechanisms of Mitochondrial Genomes
Replication…

HOW DOES IT OCCUR IN MOST ANIMALS?

A
  1. *two strands have different densities
    1. *H - heavy strand (contains most genes)
    2. *L - light strand
  2. *formation of a D (displacement) loop
  3. *both strands are not always replicated synchronously
  4. *semi-conservative
  5. *dependent on ENZYMES encoded by the NUCLEAR GENOME
24
Q

Basic Genetic Mechanisms of Mitochondrial Genomes

‘Transcription’… HOW IN human mtDNA = 9

A
  1. *D loop region contains promoters for H
    and L strands
  2. *transcription of two strands is in
    opposite directions
  3. *large transcripts made from each
    strand, later cleaved into individual
    RNAs
  4. *many genes lack complete stop codon
  5. *end in U or UA, with addition of
    poly(A) tail completing stop codon
  6. *multiple promoters in plants, fungi and other protists
  7. *no 5’ cap on mRNA
  8. *poly(A) tails on animal mitochondrial
    mRNAs
  9. *intron splicing
25
Q

UNDERSTANDING…. RNA EDITING IN MITOCHONDRIA = 5

A
  1. *changes the nucleotide sequence of transcripts
  2. *creation of an open reading frame
  3. *creation of translation start and stop codons
  4. *typically changes the amino acid sequence relative to that predicted by the DNA
  5. *may involve > half of the nucleotides in the mature transcript being changed
26
Q

RNA Editing in Mitochondria: GUIDE RNA-MEDIATED EDITING PROCESS = 12

A
  1. *guide (g)RNA
    1. *40-80 nucleotides
  2. *three regions
    1. *5’-end = anchor – directs gRNA to
      target transcript that will be edited
    2. *middle region = editing region –
      determines where Us will be inserted
      by terminal uridylyl transferase
      (TUTase)
    3. *3’-end = polyU region
  3. *editing is in a 3’ to 5’ direction
  4. *multiple gRNAs may be involved to completely edit a transcript
  5. *may also lead to deletion of U

10.*endonuclease cleavage of transcript

  1. *exonuclease removal of U
  2. *RNA ligase
27
Q

RNA Editing in Mitochondria: GUIDE RNA-MEDIATED EDITING PROCESS ….IMPORTANT DIAGRAM

28
Q

RNA Editing in Mitochondria:

‘non-gRNA-mediated editing’ = 5

A
  1. *substitution editing
  2. *sequences of edited RNA and its gene are colinear but not identical
  3. *nucleotide removal and replacement reactions
  4. *deaminase reactions, i.e. C-to-U editing
  5. *mechanisms not all known
29
Q

Basic Genetic Mechanisms of Mitochondrial Genomes …‘TRANSLATION’ = 3

A

Translation

  1. *AUG start codon codes for N-formylmethionine (as in eubacterial translation)
  2. *more wobble in human mitochondrial translation than cytoplasmic translation – any of the four nucleotides recognised in the third position (fewer tRNAs needed for
    translation
30
Q

Basic Genetic Mechanisms of Mitochondrial Genomes: ‘NONUNIVERSAL CODONS FOUND IN mtDNA…TABLE

A

SLIDE 25

CODNS, UNIVERSAL CODE … AND mtDNA

31
Q

Structure of Chloroplast Genomes: ‘cpDNA’ = 13

A

cpDNA

  1. *complexed with histone-like proteins
    1. *nucleoids
  2. *circular, linear, both forms
  3. *recombination between copies
  4. *120-160 kb
  5. *in most plants divided into 4 regions
    1. *large single-copy (LSC) region
    2. *small single copy (SSC) region
      • 2 inverted repeat (IR) regions
  6. *coding capacity
    1. *50-200 protein-coding genes
      12. *often in prokaryotic arrangement
      1. *on both strands
32
Q

Structure of Chloroplast Genomes TABLE… DIAGRAM…

33
Q

Basic Genetic Mechanisms of Chloroplast Genomes….’ REPLICATION’ = 2

A
  1. *little known
  2. *electron microscopy suggests D loop
    formation (see mtDNA replication), then
    rolling circle type mechanism
34
Q

Basic Genetic Mechanisms of Chloroplast Genomes: ‘TRANSCRIPTION’ = 8

A
  1. *arrows show the direction of transcription of the two strands
  2. *multiple promoters essentially identical to those of eubacteria
  3. *genes transcribed as groups
  4. *no 5’ cap on mRNA
  5. *no poly(A) tail on mRNA
  6. *intron splicing
  7. *RNA editing
    8. *C-to-U transitions

DIAGRAM ON SLIDE 28

35
Q

Basic Genetic Mechanisms of Chloroplast Genomes: ‘TRANSLATION’ = 4

A
  1. *AUG start codon codes for ‘N’ -formylmethionine (as in eubacterial
    translation)
  2. *initiation, elongation and termination factors essentially eubacterial
  3. *Shine-Dalgarno sequence (ribosome
    binding) often present
  4. *universal code used
36
Q

Comparison of Eukaryotic, Eubacterial and Organellar Basic Genetic Mechanisms;

TABLE

37
Q

UNDERSTANDING…Cytoplasmic Male Sterility – Mitochondrial and Nuclear
Genome Interaction = 7

A
  1. Cytoplasmic male sterility (CMS)
  2. *controlled by plant mitochondrial genomes
  3. *maternally transmitted
  4. *inability to produce functional pollen
  5. *except in male reproductive properties, plants are usually phenotypically normal
  6. *genes for the trait have been found in many different plant species
    1. *encode a variety of proteins
38
Q

UNDERSTANDING ‘CMS GENES’ = 6

A
  1. *at least 14 mitochondrial genes responsible for CMS identified
  2. *most are gain of function mutations
  3. *chimeric genes resulting from recombination events between mitochondrial genomes
  4. *often contain:
    1. *parts of genes encoding ATP
      synthase (red) and cytochrome
      oxidase (yellow) subunits
    2. *sequences of unknown origin
      (shades of blue)

DIAGRAM ON SLIDE 32

39
Q

CMS Phenotypes = 6

A
  1. *’ altered floral morphology’
    …..2. *reduction or absence of male
    reproductive parts (stamen, anthers, pollen grains)
  2. *‘Homeotic CMS phenotypes’
    …4. *decreased expression of nuclear-encoded homeotic genes, e.g. MADSbox transcription factors involved in
    floral development
  3. ’ degenerative CMS phenotypes’
    ….6.    anther, pollen degradation
40
Q

Restoration of CMS Plant Fertility = 5

A
  1. *fertility-restoration (Rf) genes
    1. *repress or neutralise genes associated with CMS
    2. nuclear encoded
      4.      often more than one locus needed
    3. *most cloned restorer genes are
      members of the pentatricopeptide-repeat (PPR) protein family
41
Q

Restoration of CMS Plant Fertility – UNDERSTANDING PPR Proteins = 4

A
  1. *large gene family in animals, plants, algae, and fungi
  2. *modular proteins
    1. *non-identical repeats of 35 (P), 36 (L) or 31(S) amino acids
  3. *involved in interactions between RNA molecules and proteins that act on them
42
Q

Restoration of CMS Plant Fertility – UNDERSTANDING PPR Proteins… DIAGRAM

A

N-terminus

Repeat Structure

C-terminus

ON SLIDE 35

43
Q

Restoration of CMS Plant Fertility – PPR Proteins… WHAT DOES IT DO? = 3

A
  1. *mechanism of restoration of fertility in CMS plants
    2. *usually product of CMS gene does not accumulate when restorer gene is present
   
     3. *may affect CMS gene expression at the level of the transcript and/or protein
44
Q

Organelle Genetics – Prospects for Biotechnology

A

Transformation of the chloroplast genome

45
Q

Organelle Genetics – Prospects for Biotechnology

Transformation of the chloroplast genome
- ADVANTAGES = 5

A

advantages OVER NUCLEAR TRANSFORMATION

  1. *high-level production of transgene
    product
  2. *highly precise integration of
    transgene(s) due to homologous
    recombination
  3. *transgene stacking in operons
  4. *no epigenetic effects
  5. *no spreading of transgenic pollen
46
Q

Organelle Genetics – Prospects for Biotechnology

Transformation of the chloroplast genome - POTENTIAL FOR: 4

A
  1. *herbicide and pathogen resistance
  2. *biopharmaceuticals
  3. *metabolic pathway modification
    4. *a second “Green Revolution”
47
Q

Organelle Genetics – Prospects for Biotechnology DIAGRAM AND PROCESS…

48
Q

Chloroplast Transformation: IMPORTANT DIAGRAM

A

TRANSFORMATION VECTOR

A, B AND C

SLIDE 38

49
Q

Chloroplast Transformation: WHY AND WHAT SO FAR? = 5

A
  1. *biolistic-mediated transformation (most common method)
    1. *introduction of foreign DNA across two membranes of chloroplast envelope
  2. *selection on medium containing the antibiotic that corresponds to the resistance marker gene in transformation
    vector
  3. *regenerate plantlets
  4. *further selection to ensure all copies of “transplastome” contain transgene
50
Q

Chloroplast Transformation – Pathogen Resistance: ‘cry’ gene = 4

A

*‘cry’ genes

  1. *encode crystal toxin proteins from Bacillus thuringiensis
  2. *multiple cry genes producing proteins that are active against different insect
    species
  3. *high levels of protein made – e.g. 45% of total leaf soluble protein
  4. *some transformed crop plants commercialised
51
Q

Chloroplast Transformation –
Biomaterials and Agronomic Traits.. TABLE

A

SLIDE 41

BIOMATERIALS AND ENZYMES ENGINEERED VIA CHLOROPLAST GENOME TOBACCO

PLASTID TRANSFORMATION, ADVANCES IN AGRONOMIC TRAITS

51
Q

Chloroplast Transformation – Pathogen Resistance… photo

A

Soybean leaves subjected to insect
bioassay
S = transplastome containing noninsecticidal gene
WT = wild type
C = transplastome containing ‘cry’ gene

SLIDE 40

52
Q

Chloroplast Transformation – Molecular “Pharming” = 2

A
  1. *low production costs, ease of scaling
    up production and high safety (no risk
    of contamination with human pathogen
    and/or endotoxins in product)
  2. *targets = antigens, antibodies
    (“plantibodies”), and antimicrobials

TABLE: PLATID TRANSFORMATION, ADVANCES IN BIOPHARMACEUTICAL

Genomics Theory (10 decks)

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