Organelle Genetics I & II (Lectures 8&9) Flashcards
(55 cards)
1
Q
Understanding Organelle DNA heredity: 7
A
- *a type of EXTRANUCLEAR inheritance
- *NON-MENDELIAN inheritance
- *UNIPARENTAL inheritance
- *typically MATERNAL
- *female contributes BULK OF CYTOPLASM TO PROGENY
- *are EXCEPTIONS; PARENTAL, BIPARENTAL
- *typically MATERNAL
7.*NO SEGREGATION RATIOS AS FOR NUCLEAR GENES
2
Q
UNDERSTANDING Chloroplast DNA Heredity = 6
A
- *VARIEGATED PLANTS
- *white regions
- *CELLS WITH MUTATION IN A GENE CODING FOR A PROTEIN INVOLVED IN CHLOROPHYLL SYNTHESIS
4. *gene is in chloroplast genome (cpDNA)
- *CELLS WITH MUTATION IN A GENE CODING FOR A PROTEIN INVOLVED IN CHLOROPHYLL SYNTHESIS
- *white regions
- *chloroplast GENES IN FLOWERS ARE SAME as those ON SUPPORTING BRANCH
- *e.g., white branch - male and female
gametes will have cpDNA with
MUTATION
3
Q
Chloroplast DNA Heredity PROCESS: 4
A
- *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)
- ZYGOTES CONTAINING BOTH TYPES OF cpDNA often show ‘CYTOPLASMIC SEGREGATION’ as they DIVIDE
4
Q
Chloroplast DNA Heredity DIAGRAM
A
SLIDE 6
5
Q
cytohets (heteroplasmons) ?
A
*cytohets (heteroplasmons)
*cells with a mixture of organelle genomes
6
Q
Cytoplasmic Segregation IN CYTOHETS = 4
A
- *cytohets (heteroplasmons)
*cells with a mixture of
organelle genomes - FOLLOWING MITOSIS*
*PROGENY WITH MIXTURE
* PROGENY WITH ONE OR OTHER —> ‘CYTOPLASMIC SEGREGATION’
- FOLLOWING MITOSIS*
- *CHANCE EVENTS
- *ORGANELLES DO NOT SEGREGATE TO POLES ALONG MITOTIC SPINDLE, ‘STOCHASTIC PARTIONING INSTEAD’
DIAGRAM IN SLIDE 7
7
Q
Cytoplasmic Inheritance in Humans: 6
A
- *number of mutations in mitochondrial genes that can cause disease
- HUMAN PEDIGREES SHOW PHENOTYPES TRANSMITTED FROM MOTHERS TO SONS AND DAUGHTERS…
- *NOT ALL COPIES OF MITOCHONDRIAL DNA (mtDNA) IN A CELL WILL HAVE THE MUTATION.
- SEVERITY OF DISEASE ASSOCIATED WITH PROPORTION OF MUTATED mtDNA INHERITED.
- *2018 (Luo et al. PNAS 115: 13039) –
EVIDENCE OF BIPARENTAL INHERITANCE of MITOCHONDRIAL DNA in HUMANS - *DEEP SEQUENCING INDICATES biparental inheritanceMORE COMMON THAN ANTICIPATED
7
Q
Cytoplasmic Inheritance in Humans
‘Myoclonic epilepsy and ragged red fibre
(MERRF) disease’
A
- *lack of muscle coordination, deafness, dementia
- *“ragged red” - muscle fibre appearance
- *single base change leading to mutation in mitochondrial tRNA(Lys)
8
Q
Cytoplasmic Inheritance in Humans:
‘Leber hereditary optic neuropathy (LHON)’
A
- SUDDEN BILATERAL BLINDNESS
- *4 mutations identified - all lead to disruption of OXIDATIVE PHOSPHORYLATION
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
- *little or NO RECOMBINATION
- *EVOLVES AT FASTER RATE than nuclear DNA (GOOD FOR STUDYING CLOSELY RELATED GROUPS)
- *1 change per mitochondrial lineage every 3800 years
- *maternally inherited
- CAN ESTIMATE THE NUMBER OF YEARS SINCE POPULATIONS HAVE BEEN SEPARATED
10
Q
Cytoplasmic Inheritance in Humans – Reconstructing Relationships Among Populations
mtDNA … ANCESTRAL RELATIONSHIPS?
MOST RECENT ANCESTOR?
A
- 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
11
Q
Division and Segregation of Organelles - Chloroplasts: 12
A
- *chloroplasts come from PRE-EXISTING chloroplasts
- *requires INTERACTION of PROKARYOTE-DERIVED and EUKARYOTE-DERIVED MACHINERIES
- *FtsZ ring
*FtsZA, FtsZB (filamentous temperature-sensitive) proteins
*bacterial cell division proteins
*form a ring inside chloroplast, lining inner membrane surface - *plastid-dividing ring (PDR)
*nanofilaments (polyglucan)
*eukaryotic origin
*form inside and outside
organelle - *dynamin ring
*dynamin-related protein
(eukaryotic membrane REmodeling GTPases)
*forms ring outside the
chloroplast - PDR and dynamin rings twist to
pinch membrane - *chloroplasts interact with cytoskeletal
components during cytokinesis - *details of segregation not known
11
Q
Division and Segregation of Organelles - Mitochondria = 6
A
- *FtsZ ring
*forms a ring inside mitochondrion, lining the inner membrane surface - *mitochondrial-dividing (MD) rings
*form inside and outside organelle
*nanofilaments (polyglucan)
*eukaryotic origin - *dynamin ring
*forms outside organelle
*eukaryotic origin - *MD and dynamin rings twist to pinch membrane
- *mitochondria interact with cytoskeletal components during cytokinesis
- *details of segregation not known
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
13
Q
Structure of Mitochondrial Genomes: 5
A
- *higher plant mtDNA exhibits high levels of recombination
- *crossing over between large repeat regions
- *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
14
Q
diagram : Structure of Mitochondrial Genomes
A
slide 15
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
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
17
Q
RECLINOMONAS
A
ANCESTRAL - GREAT NUMBER OF GENES IN MITOCHONDRIAL GENOME
18
Q
PLASMODIUM
A
HIGHLY DERVIED - FEWEST GENES
19
Q
Basic Genetic Mechanisms of Mitochondrial Genomes: 3
A
- *genes on both strands
- *“machinery” for replication, transcription & translation encoded by mtDNA and nuclear genome
- *gene products from both genomes are required for functional organelles
20
Q
Basic Genetic Mechanisms of Mitochondrial Genomes IMPORTANT DIAGRAM
A
SLIDE 18
21
Q
Basic Genetic Mechanisms of Mitochondrial Genomes
‘ Replication’ =
A
- *mtDNA synthesised throughout cell cycle, NOT COORDINATED with synthesis of nuclear DNA
- *which molecules are replicated is RANDOM
- *within an organelle some molecules replicated MULTIPLE TIMES, OTHER NOT REPLICATE r
22
Q
Basic Genetic Mechanisms of Mitochondrial Genomes
‘Replication’ IMPORTANT DIAGRAM
A
SLIDE 19
23
Basic Genetic Mechanisms of Mitochondrial Genomes
Replication...
HOW DOES IT OCCUR IN MOST ANIMALS?
1. *two strands have different densities
2. *H - heavy strand (contains most genes)
3. *L - light strand
4. *formation of a D (displacement) loop
5. *both strands are not always replicated synchronously
6. *semi-conservative
7. *dependent on ENZYMES encoded by the NUCLEAR GENOME
24
Basic Genetic Mechanisms of Mitochondrial Genomes
'Transcription'... HOW IN human mtDNA = 9
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
UNDERSTANDING.... RNA EDITING IN MITOCHONDRIA = 5
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
RNA Editing in Mitochondria: GUIDE RNA-MEDIATED EDITING PROCESS = 12
1. *guide (g)RNA
2. *40-80 nucleotides
3. *three regions
4. *5’-end = anchor – directs gRNA to
target transcript that will be edited
5. *middle region = editing region –
determines where Us will be inserted
by terminal uridylyl transferase
(TUTase)
6. *3’-end = polyU region
7. *editing is in a 3’ to 5’ direction
8. *multiple gRNAs may be involved to completely edit a transcript
9. *may also lead to deletion of U
10.*endonuclease cleavage of transcript
11. *exonuclease removal of U
12. *RNA ligase
27
RNA Editing in Mitochondria: GUIDE RNA-MEDIATED EDITING PROCESS ....IMPORTANT DIAGRAM
SLIDE 23
28
RNA Editing in Mitochondria:
'non-gRNA-mediated editing' = 5
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
Basic Genetic Mechanisms of Mitochondrial Genomes ...'TRANSLATION' = 3
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
Basic Genetic Mechanisms of Mitochondrial Genomes: 'NONUNIVERSAL CODONS FOUND IN mtDNA...TABLE
SLIDE 25
CODNS, UNIVERSAL CODE ... AND mtDNA
31
Structure of Chloroplast Genomes: 'cpDNA' = 13
cpDNA
1. *complexed with histone-like proteins
2. *nucleoids
3. *circular, linear, both forms
4. *recombination between copies
5. *120-160 kb
6. *in most plants divided into 4 regions
7. *large single-copy (LSC) region
8. *small single copy (SSC) region
9. * 2 inverted repeat (IR) regions
10. *coding capacity
11. *50-200 protein-coding genes
12. *often in prokaryotic arrangement
13. *on both strands
32
Structure of Chloroplast Genomes TABLE... DIAGRAM...
SLIDE 27
33
Basic Genetic Mechanisms of Chloroplast Genomes....' REPLICATION' = 2
1. *little known
2. *electron microscopy suggests D loop
formation (see mtDNA replication), then
rolling circle type mechanism
34
Basic Genetic Mechanisms of Chloroplast Genomes: 'TRANSCRIPTION' = 8
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
Basic Genetic Mechanisms of Chloroplast Genomes: 'TRANSLATION' = 4
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
Comparison of Eukaryotic, Eubacterial and Organellar Basic Genetic Mechanisms;
TABLE
SLIDE 30
37
UNDERSTANDING...Cytoplasmic Male Sterility – Mitochondrial and Nuclear
Genome Interaction = 7
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
7. *encode a variety of proteins
38
UNDERSTANDING 'CMS GENES' = 6
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:
5. *parts of genes encoding ATP
synthase (red) and cytochrome
oxidase (yellow) subunits
6. *sequences of unknown origin
(shades of blue)
DIAGRAM ON SLIDE 32
39
CMS Phenotypes = 6
1. *' altered floral morphology'
.....2. *reduction or absence of male
reproductive parts (stamen, anthers, pollen grains)
3. *'Homeotic CMS phenotypes'
...4. *decreased expression of nuclear-encoded homeotic genes, e.g. MADSbox transcription factors involved in
floral development
5. *' degenerative CMS phenotypes'
....6.*anther, pollen degradation
40
Restoration of CMS Plant Fertility = 5
1. *fertility-restoration (Rf) genes
2. *repress or neutralise genes associated with CMS
3. *nuclear encoded
4.*often more than one locus needed
5. *most cloned restorer genes are
members of the pentatricopeptide-repeat (PPR) protein family
41
Restoration of CMS Plant Fertility – UNDERSTANDING PPR Proteins = 4
1. *large gene family in animals, plants, algae, and fungi
2. *modular proteins
3. *non-identical repeats of 35 (P), 36 (L) or 31(S) amino acids
4. *involved in interactions between RNA molecules and proteins that act on them
42
Restoration of CMS Plant Fertility – UNDERSTANDING PPR Proteins... DIAGRAM
N-terminus
Repeat Structure
C-terminus
ON SLIDE 35
43
Restoration of CMS Plant Fertility – PPR Proteins... WHAT DOES IT DO? = 3
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
Organelle Genetics – Prospects for Biotechnology
Transformation of the chloroplast genome
45
Organelle Genetics – Prospects for Biotechnology
Transformation of the chloroplast genome
- ADVANTAGES = 5
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
Organelle Genetics – Prospects for Biotechnology
Transformation of the chloroplast genome - POTENTIAL FOR: 4
1. *herbicide and pathogen resistance
2. *biopharmaceuticals
3. *metabolic pathway modification
4. *a second “Green Revolution”
47
Organelle Genetics – Prospects for Biotechnology DIAGRAM AND PROCESS...
SLIDE 37
48
Chloroplast Transformation: IMPORTANT DIAGRAM
TRANSFORMATION VECTOR
A, B AND C
SLIDE 38
49
Chloroplast Transformation: WHY AND WHAT SO FAR? = 5
1. *biolistic-mediated transformation (most common method)
2. *introduction of foreign DNA across two membranes of chloroplast envelope
3. *selection on medium containing the antibiotic that corresponds to the resistance marker gene in transformation
vector
4. *regenerate plantlets
5. *further selection to ensure all copies of “transplastome” contain transgene
50
Chloroplast Transformation – Pathogen Resistance: 'cry' gene = 4
*'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
Chloroplast Transformation –
Biomaterials and Agronomic Traits.. TABLE
SLIDE 41
BIOMATERIALS AND ENZYMES ENGINEERED VIA CHLOROPLAST GENOME TOBACCO
PLASTID TRANSFORMATION, ADVANCES IN AGRONOMIC TRAITS
51
Chloroplast Transformation – Pathogen Resistance... photo
Soybean leaves subjected to insect
bioassay
S = transplastome containing noninsecticidal gene
WT = wild type
C = transplastome containing 'cry' gene
SLIDE 40
52
Chloroplast Transformation – Molecular “Pharming” = 2
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
