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