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EXTRANUCLEAR INHERITANCEEXTRANUCLEAR INHERITANCE
FUNCTIONS
PHOTOSYNTHESIS
nCO2 + 2nH2O (CH2O)n + nO2
[sun energy]
PRODUCTION OF CHEMICAL ENERGY
(CH2O)n + nO2 nCO2 + 2n H2O
energy
ATP + heat production
MITOCHONDRIA CHLOROPLAST
Production of ATP and NADPH
Euglena gracilis
cpmt
Light-independent reactions
Light-dependent reactions
mtDNA
Mitochondrial genome :
Human 37 genes
2 rRNA
22 tRNA
13 subunits of respiratory chain
complex (tot. ≅ 80 proteins)
COX genes (Cytochrome oxidase)
ND genes (NADH dehydrogenase)
Yeast mitochondrial DNA ≅ 85Kbp
Example of a multimeric protein: complex IV
(Cytocrome Oxidase ) : 3 subunits coded by mitochondrial genes, 10 subunitscoded by nuclear genes.
Yeast mitochondrial DNA ≅ 85Kbp
Yeast Genome 5 times larger, but only 6 additional genes
(tot. 43)
Ex. RuBisCOuBisCO (ribulose-1,5-biphosphate carboxylase-oxygenase) –About 30-50% of the soluble proteins in leaves [FIXATION of CO2]
8 subunits rbcL (plastid encoded)
8 subunits rbcS (nuclear encoded)
InvertedRepeats
psb → photosynthesis related genes
CLONING- DNA Cloning → Insertion of DNA fragments into suitable DNA vectors
- Embryo Cloning → Production of versatile stem cells, which can differentiate into various types of cells, for medical purposes
- Reproductive Cloning → Replacing the genetic material of an egg with that of anadult cell, it should be possible to create an exact copy of another living organism
Genetic Production of mammal clones. [Roslin Institute – Edinburgh -1997]
Nucleus from a 6 years old Finn Dorset sheep
Enucleated egg from a Scottish Blackface sheep
Reproductive cloning is an inefficient (< 1% success) and error-prone process.
Many cloned embryos are miscarried; others have abnormalities.
However, it was demonstrated for the first time that highly differentiated cells are still TOTIPOTENT
What about mitochondria ?
GENOME SIZE IN:
MITOCHONDRIA CHLOROPLAST
Notes:
cp algal genomes have the largestsize
cp (~ 120 genes); mt (~ 40 genes)
cp (~ 50 proteins); mt (< 20)
cp synthesize their own RNA pol.
absence of histons
Comparison between nuclear, eubacterial, mt and cp genomes
Endosymbiotic Theory (Lynn Margulis 1967)
Alternative Theory – Initiation of Translation is different from both eubacteriaand eucaryotes
Food for thought … about symbiosis
N° of cells N° of cells
Escherichia coli, a member of human flora
Consider that:
- less than 10% of microbiota can be cultivated)
- bacterial cells exceeds more than 10 times the number of human cells!
The Endosymbiotic Theory postulates that:
- mitochondria are the result of endocytosis of aerobic bacteria- chloroplasts are the result of endocytosis of photosynthetic bacteria(cyanobacteria)- in both cases by large anaerobic bacteria (?) who would not otherwise beable to exist in an aerobic environment. - this arrangement became a mutually beneficial relationship for both cells(symbiotic).
There are several examples of bacteria (small genomes) living inside eucaryoticorganisms
Ancestral prokaryoteAnaerobic bacteria
Multiple copies of mitochondria/cell
Multiple copies of mt genome / mitochondria
Liver cells contain up to 10.000 copies of the mt genome(approx. 5-10 copies of mtDNA/mitochondria)
Inheritance mechanisms and transmission of characters differ significantlybetween nuclear and organelle genomes.
- Mitocondria and Plastids reproduction is not connected with cell division
- organelles are NOT distributed equally between daughter cells
- Meiosis has no influence on organelle genomes;
no genetic reshuffling takes palce
- Mendel’ laws cannot be applied
- genes present in the mitochondrial DNA (and their mutations) are inherited:
exclusively from maternal lineage, from egg-cell (monoparentalinheritance, as in H. sapiens) consequence, very low level of recombination
from both cells which merge to produce a zygote (biparentalinheritance, as in Yeast, and some plants)
SEGREGATION OF MITOCHONDRIA
THRESHOLD EFFECT
Heteroplasmic cells
When variants of mitochondrialDNA coexist in the cytoplasm of the same cell.
Homoplasmy versus Heteroplasmy
“petite” mutants in yeast (Boris Ephrussi, 1950)
Analysis of mt DNA
Very large deletions of mtDNA were found.
In some extreme cases mtDNA wascompletely absent
Cells are unable to produce enzymes requiredfor aerobic respiration
These mutant cells can produce chemical energyONLY via ANAEROBIC respiration (Glycolysis ) and this process is much LESS EFFICIENT
than Oxidative phosphorylation Weak growth, very little colonies
TRANSMISSION OF MITOCHONDRIA - RELATED DISEASES
SEX-LINKED DISEASES
Male progeny → HEALTHY Female progeny → +/- AFFECTED
Some diseases appear phenotypically onlywhen the threshold is reached.
They can be monitored genetically !
It is possible to predict the genoptypeof the offspring
Note:
≅1016 mtDNA molecules are present within a human
Human mitochondrial genome is subjected to a mutation rate approx. 10-17 times higher than nuclear genome (O2 metabolism ?)
~ 150 mutations correlated with diseases have beeen identified
(~ 50 in enzyme-coding genes; ~ 100 in tRNA genes “hot-spots”)
Low level of mutation
High level of mutation
Intermediate level ofmutation
tRNA Processing
CCA addition
Aminoacylation
5’ end Cleavage 3’ end Cleavage
Mitochondrial Theory of Ageing
This theory proposes that accumulation of mutations in mitochondrialDNA leads to progressive bioenergy deficiency, cellular damage, degeneration and eventually death.
deafness
MELAS Mitochondrial Encephalomyopathy with lactic acidosis and stroke-like episodes
MERRF Myoclonus Epylepsy and Ragged-Red Fibers
Pathologies related to mutations of mtDNA
*
* Abnormality of combined ocular motility. Paralysis of the muscles which are responsible for the eye movement → Due to deletion (1-9 Kbp)
Always heteroplasmic
Nature (1987) 325: 31-36
Genealogical tree of human mtDNA
The common ancestral mtDNA linksmtDNA types that have diverged around140.000 – 290.000 years ago
OUT-OF-AFRICA HYPOTHESYS
mt DNA is maternally inherited and does not recombine, so it is a toolfor relating individuals. Mutations accumulate faster than in the nucleus → it evolves quickly.
In human genetics, a human mitochondrial DNA haplogroup is a haplogroup defined by differences in human mitochondrial DNA.
Haplogroup: a large set of HAPLOTYPES (haploid genotypes)
A combination of alleles at multiple linked loci that are transmitted together. Genetic constitution of one member of the pair of alleles for each locus
These haplogroups trace the matrilineal inheritance of modernhumans back to human origins in Africa (some 200.000 years ago) and the subsequent spread across the globe. They are termed A, B, C, L0, L1, L2, L3, etc.
Haplotypes are combinations of gene variants, or SNPs, thatare likely to be inherited together within the samechromosomal region.
In this example, an original haplotype (top) evolved over timeto create four newer haplotypes that each differ by a fewnucleotides (red).
Common Ancestor
Africa presents the most complex genetic picture of any continent;
the diversity of genetic markers indicates that Africa was the earliest home of modern humans.
GENO-GRAPHIC Project
Collection of DNA in cheek swabs and blood samplesfrom hundreds ofindigenous groups.
Targets:
-Y chromosome (travelsintact from father to son)
- mtDNA which a motherpasses to her offspring
Over generations, smallharmless mutationsaccumulate on these twotargets: these geneticmarkers constitute a historybook.
As ancient human populations migratedout of Africa, entering new lands, theyaccumulated different patterns ofmarkers that reflect that history.
Each individual today retains suchpattern.
Source: National Geographic
A phylogenetic tree is a specific type of cladogram where the branch lengths are proportional to the predicted or hypothetical evolutionary time between organisms or sequences.
Cladograms are branched diagrams, similar in appearance to family trees, that illustrate patterns of relatedness.
Example
Sequence A: 5’ GGAACCTT
Sequence B: 5’ GGAACCTA
Sequence C: 5’ GCAACCTG
Sequence D: 5’ GCAACCTC
A B C D
Rooted Tree
Bioinformaticians produce cladograms representing relationships between sequences, either DNA sequences or amino acid sequences. However, cladograms can rely on many types of data to show the relatedness of species. In addition to sequence homology information, comparative embryology, fossil records and comparative anatomy are all examples of the types of data used to classify species into phylogenic taxa.
So, it is important to understand that the cladograms generated by bioinformatic tools are primarily based on sequence data alone. These cladograms can be very powerful as a predictor of the relatedness of species.
Phylogenetic trees are genealogical trees (graphical representations) whichare built up with information gained from the comparison of the DNA sequences (or amino acid sequences of a protein like cytochrome C, whichoccurs throughout the living matter).
Due to Darwinian Evolution, the DNA sequence (or protein sequence) hasslightly different features for each of the species.
A taxon is a Biological Unit(species, genus, family)
The points at which branches separate are called nodes and represent hypothetical ancestors of the descendant clades (more technical language, clades being a group of taxa) of that node.
Pairs of taxa or clades are said to be sisters (so A and B are sisters, as are E and F). Sister taxa of course share a recent common ancestor at the node that joins them together.
Often the base of the tree (typically a single taxon, but sometimes more) is called the outgroup. This is the taxon used to act as a basis for the analysis that produced the tree; everything else is part of the ingroup.
(Underground map)
The tree on the right (2) is identical to tree N.1, but a few branches have been rotated around the nodes.
It “looks” different, but it is not, all the relationships are the same –the branch supporting E and F evolves before A-D, and C appears before A and B, but E and F appear to be the most advanced.
1. The aadA (amino glycosideadenyltransferase ) gene istethered to a plastid promoter;
2. Transgene integration occurs bytwo homologous recombinationevents;
3. Any transgene of interest can beco-incorporated by physicallylinking it to the aadA marker gene
4. The transformation vector is shotby gene gun into living leaf cells on the surface of gold particles;
5. Small leaf particles are exposed toa regeneration medium containingspectinomycin (a plastid inhibitor);
6. Several rounds of regenerationunder antibiotic selection are necessary to convert heteroplasmicinto homoplasmic genomes;
7. Homoplasmic shoots are rooted.
Targeting of a transgene to a non-codingintergenic region of the plastid genome(ptDNA)
APPLICATIONS OF BIOLISTIC PROCEDURE
- Improvement of Photosynthesisthrough RuBisCO engineering. Very slow enzyme !
- Synthesis of β-carotene (provitamin A)
- Production of rare AA (Trp)
- Controlled ripening
- Fungal/viral resistance
- Herbicide tolerance
- Production of trehalose (sugar)
Microprojectiles coated with DNA
TREHALOSE
Osmoprotective agent used as excipientfor the preservation of pharmaceuticals, vaccines, organs for surgical transplants.
Also effective as a bioprotector in dehydration and freezing processes fordried and processed food, due to high water retention capabilities.
gluc
fruct
gluc gluc
Introduction of the yeast trehalose phosphate synthase gene TPS1 intothe tobacco chloroplast genome resulted in ≅ 20 fold increase in trehalose accumulation.
JILIN AGRICULTURAL UNIVERSITY 吉林农业大学
Development of methods for particle-mediated genetic transformation of tobacco
badH NOS poly(A)35S Pro
beta-glucuronidase
Helium pressure gauge
Fire switch
Vac/Vent/Hole switch
Power switch
Bombardment chamber doorDisk retaining cap
Vacuum gauge
Microcarrier launchassembly
Target shelf
Vacuum/Vent Rate Control Valves
Biolistic® PDS-1000/He device
Biolistic® Gene Gun
Results
Two of the five callus samples transformed with nanoparticles clearly showed blue spots.
Blue spot
(C)(B)(A)
1 day 14 days 24 days
Application:
- a tool for the design and the synthesis of complete mitochondriachromosomes, which may be expressed once installed in mitochondria
- could be used to restore respiratory competence to mammaliancells with mitochondrial deficiencies
The first synthetic genome of anorganelle to be made
Nature Methods (2010) 7: 901-903