Mitochondrial DNA

John 1:12

12

But as many as received him, to them gave he power to become the sons of God, even to them that believe on his name:

©2001 Timothy G. Standish

Endosymbiosis and the

Origin of Eukaryotes:

Are mitochondria really just bacterial symbionts?

Timothy G. Standish, Ph. D.


Outline

Mitochondria A very brief overview

Endosymbiosis Theory and evidence

Archaezoa Eukaryotes lacking mitochondria

Gene expression Mitochondrial proteins coded in the nucleus

Mitochondrial genetic codes

Gene transport Mitochondria to nucleus

Conclusions


Mitochondria

Mitochondria are organelles found in most eukaryotic organisms.

The site of Krebs cycle and electron transport energy producing processes during aerobic respiration

Are inherited only from the mother during sexual reproduction in mammals and probably all other vertebrates.

Because of their mode of inheritance genetic material found in mitochondria appears to be useful in determining the maternal lineage of organisms.


Matrix

Inter membrane space

Mitochondria

Outer membrane

Inner membrane mtDNA


Extranuclear DNA

Mitochondria and chloroplasts have their own DNA

This extranuclear DNA exhibits non-Mendelian inheritance

Recombination is known between some mt and ctDNAs

Extranuclear DNA may also be called cytoplasmic DNA

Generally mtDNA and ctDNA is circular and contains genes for multimeric proteins, some portion of which are also coded for in the nucleus

Extranuclear DNA has a rate of mutation that is independent of nuclear DNA

Generally, but not always, all the RNAs needed for transcription and translation are found in mtDNA and ctDNA, but only some of the protein genes


mtDNA

Mitochondrial DNA is generally small in animal cells, about 16.5 kb

In other organisms sizes can be more than an order of magnitude larger

Plant mtDNA is highly variable in size and content with the large Arabidopsis mtDNA being 200 kb.

The largest known number of mtDNA protein genes is 97 in the protozoan Riclinomonas mtDNA of 69 kb.

“Most of the genetic information for mitochondrial biogenesis and function resides in the nuclear genome, with import into the organelle of nuclear DNA-specified proteins and in some cases small RNAs.” (Gray et al .,1999)


Endosymbiosis


Origin of Eukaryotes

Two popular theories presupposing naturalism seek to explain the origin of membrane bound organelles:

1 Endosymbiosis to explain the origin of mitochondria and chloroplasts (popularized by Lynn Margulis in 1981)

2 Invagination of the plasma membrane to form the endomembrane system






Mitochondria






Mitochondria

Endoplasmic

Reticulum

Chloroplast

Nucleus

Golgi

Body






Endoplasmic Reticulum

Mitochondria

Nucleus

Chloroplast

Golgi Body


How Mitochondria Resemble Bacteria

Most general biology texts list ways in which mitochondria resemble bacteria. Campbell et al.

(1999) list the following:

Mitochondria resemble bacteria in size and morphology.

They are bounded by a double membrane: the outer thought to be derived from the engulfing vesicle and the inner from bacterial plasma membrane.

Some enzymes and inner membrane transport systems resemble prokaryotic plasma membrane systems.

Mitochondrial division resembles bacterial binary fission

They contain a small circular loop of genetic material

(DNA). Bacterial DNA is also a circular loop.

They produce a small number of proteins using their own ribosomes which look like bacterial ribosomes.

Their ribosomeal RNA resembles eubacterial rRNA.


How Mitochondria Don’t

Resemble Bacteria

Mitochondria are not always the size or morphology of bacteria:

– In some Trypanosomes (i.e., Trypanosoma brucei ) mitochondria undergo spectacular changes in morphology that do not resemble bacteria during different lifecycle stages

(Vickermann, 1971)

– Variation in morphology is common in protistans,

“Considerable variation in shape and size of the organelle can occur.” (Lloyd, 1974, p 1)

Mitochondrial division and distribution of mitochondria to daughter cells is tightly controlled by even the simplest eukaryotic cells


How Mitochondria Don’t

Resemble Bacteria

Circular mtDNA replication via D loops is different from replication of bacterial DNA (Lewin, 1997, p 441).

mtDNA is much smaller than bacterial chromosomes.

Mitochondrial DNA may be linear, examples include:

Plasmodium, C. reinhardtii, Ochromonas, Tetrahymena ,

Jakoba (Gray et al., 1999).

Mitochondrial genes may have introns which eubacterial genes typically lack (these introns are different from nuclear introns so they cannot have come from that source) (Lewin, 1997 p 721, 888).

The genetic code in many mitochondria is slightly different from bacteria (Lewin, 1997).


Archaezoa


Giardia

A “Missing Link”?

The eukaryotic parasite Giardia has been suggested as a “missing link” between eukaryotes and prokaryotes because it lacks mitochondria (Friend, 1966; Adam, 1991) thus serving as an example of membrane invagination but not endosymbiosis

Giardia also appears to lack smooth endoplasmic reticulum, peroxisomes and nucleoli (Adam, 1991) so these must have either been lost or never evolved


A Poor “Missing Link”

As a “missing link” Giardia is not a strong argument due to its parasitic life cycle which lacks an independent replicating stage outside of its vertebrate host

– Transmission is via cysts excreted in feces followed by ingestion

– As an obligate parasite, to reproduce, Giardia needs other more derived (advanced?) eukaryotes

Some other free-living Archaezoan may be a better candidate


Origin of

Giardia

Giardia and other eukaryotes lacking mitochondria and plastids (Metamonada, Microsporidia, and Parabasalia ) have been grouped by some as “Archaezoa” (Cavalier-

Smith, 1983; Campbell et al ., 1999 p 524-6)

This name reflects the belief that these protozoa split from the group which gained mitochondria prior to that event.

The discovery of a mitochondrial heat shock protein

(HSP60) in Giardia lamblia (Soltys and Gupta, 1994) has called this interpretation into question.

Other proteins thought to be unique to mitochondria,

HSP70 (Germot et al ., 1996), chaperonin 60 (HSP60)

(Roger et al ., 1996; Horner et al ., 1996) and HSP10 (Bui et al.

, 1996) have shown up in Giardia ’s fellow

Archaezoans


Origin of Archaezoa

The authors who reported the presence of mitochondrial genes in amitochondrial eukaryotes all reinterpreted prevailing theory in saying that mitochondria must have been present then lost after they had transferred some of their genetic information to the nucleus.

The hydrogenosome, a structure involved in carbohydrate metabolism found in some Archaezoans (Muller, 1992), is now thought to represent a mitochondria that has lost its genetic information completely and along with that loss, the ability to do the Krebs cycle (Palmer, 1997).

Alternative explanations include transfer of genetic material from other eukaryotes and the denovo production of hydrogenosomes by primitive eukaryotes.


Origin of Archaezoa:

Mitochondrial Acquisition



Gene Transfer and Loss mtGenes

Lost genetic material



Option 1 - Mitochondrial Eukaryote Production



Option 2 - Mitochondrial DNA Loss/

Hydrogenosome production

Hydrogenosome



Option 2A - Mitochondria/Hydrogenosome Loss


Gene Transport


“All in all then, the host nucleus seems to be a tremendous magnet, both for organellar genes and for endosymbiotic nuclear genes.”

Palmer, 1997


Steps in Mitochondrial Acquisition:

The Serial Endosymbiosis Theory

Fusion of Rickettsia with either a nucleus containing Archaezoan or an archaebacterium

Rickettsia

Host Cell

Primitive eukaryote

DNA reduction/transfer to nucleus

Ancestral eukaryote

(assuming a nucleus)


Steps in Mitochondrial Acquisition:

The Hydrogen Hypothesis

Hydrogen producing proteobacterium

Fusion of proteobacterium with an archaebacterium

Hydrogen requiring archaebacterium

DNA reduction/transfer nucleus production

Ancestral eukaryote

With nucleus containing both archaebacterium and proteobacterium genes


Bacteria

Phylogeny

Microsporidia, and Parabasalia Metamonada mtDNA loss

Hydrogenosome/ mitochondria loss mtDNA loss

Eukaryota Bacteria

Gene transfer

Cell fusion

Origin of Life


Timing of Gene Transfer

Because gene transfer occurred in eukaryotes lacking mitochondria, and these are the lowest branching eukaryotes known:

Gene transfer must have happened very early in the history of eukaryotes.

The length of time for at least some gene transfer following acquisition of mitochondria is greatly shortened.

No plausible mechanism for movement of genes from the mitochondria to the nucleus exists although intraspecies transfer of genes is sometimes invoked to explain the origin of other individual nuclear genes.


Gene

Expression


Cytoplasmic Production of

Mitochondrial Proteins

Mitochondria produce only a small subset of the proteins used in the Krebs cycle and electron transport. The balance come from the nucleus

As mitochondrial genomes vary spectacularly between different groups of organisms, some of which may be fairly closely related, if all came from a common ancestor, different genes coding for mitochondrial proteins must have been passed between the nucleus and mitochondria multiple times


The Unlikely Movement of Genes

Between Mitochondria and the Nucleus

Movement of genes between the mitochondria and nucleus seems unlikely for at least two reasons:

1 Mitochondria do not always share the same genetic code with the cell they are in

2 Mechanisms for transportation of proteins coded in the nucleus into mitochondria seem to preclude easy movement of genes from mitochondria to the nucleus


Protein Production

Mitochondria and Chloroplasts

Cytoplasm

Nucleus

Export

G AAAAAA

Mitochondrion Chloroplast


Protein Production

Mitochondria and Chloroplasts

Cytoplasm

Nucleus

Mitochondrion Chloroplast


Protein Production

Mitochondria

Outer membrane

Inner membrane

Matrix



Protein Production

Mitochondria

P +ADP

ATP

Leader sequence binding receptor

Outer membrane

ATP

P +ADP

Inner membrane

Inter membrane

Matrix

Protein Production

Mitochondria


Outer membrane

Peptidease cleaves off the leader

Inner membrane

Inter membrane

Matrix

Protein Production

Mitochondria


Outer membrane

Inner membrane

Inter membrane

Matrix

Protein Production

Mitochondria


Outer membrane

Inner membrane

Inter membrane

Matrix

Protein Production

Mitochondria


Outer membrane

Matrix

Hsp60

Chaperones

Hsp60

Inner membrane

Inter membrane

Protein Production

Mitochondria


Outer membrane

Matrix

Mature protein

Inner membrane

Inter membrane

M

L

L

S

I

S

Nonpolar

F

F

K

Q

P

A

T

R

Polar

C

S

S

T

R

L

Y

P

L

Yeast Cytochrome C

Oxidase Subunit IV Leader

First 12 residues are sufficient for transport to the mitochondria

Neutral Non-polar

Polar

Basic

Acidic

ML S L R QS I R FF K PA T R T L CSS R Y LL

This leader does not resemble other eukaryotic leader sequences, or other mtProtein leader sequences.

Probably forms an a helix

This would localize specific classes of amino acids in specific parts of the helix

There are about 3.6 amino acids per turn

Yeast Cytochrome C1 Leader

Charged leader sequence signals for transport to mitochondria

First cut

MF SN L S KR WA Q R T L S K T L K GS K S AA GT A TSY F E -

K LV T A G VAAA G I T A ST LL Y A N S L T A G A--------------

Uncharged second leader sequence signals for transport

Second cut across inner membrane into the intermembrane space

Cytochrome c functions in electron transport and is thus associated with the inner membrane on the

Neutral Non-polar

Polar

Basic

Acidic intermembrane space side

Cytochrome c1 holds an iron containing heme group and is part of the B-C1 (III) complex

C1 accepts electrons from the Reiske protein and passes them to cytochrome c


Protein Production

Mitochondria

Outer membrane

Inner membrane

Matrix



Protein Production

Mitochondria

P +ADP

ATP


Outer membrane

ATP

P +ADP

Peptidease cleaves off the leader

Inner membrane

Inter membrane

Matrix

Protein Production

Mitochondria


Outer membrane

Inner membrane

Inter membrane

Matrix

Protein Production

Mitochondria


Outer membrane

Inner membrane

Inter membrane

Matrix

Protein Production

Mitochondria


Outer membrane

Inner membrane

Inter membrane

Matrix

Protein Production

Mitochondria


Outer membrane

Inner membrane

Inter membrane

Matrix

Protein Production

Mitochondria


Outer membrane

Inner membrane

Peptidease cleaves off the second leader

Inter membrane

Matrix

Protein Production

Mitochondria


Outer membrane

Inner membrane

Inter membrane

Matrix

Protein Production

Mitochondria


Outer membrane

Inner membrane

Inter membrane

Matrix

Protein Production

Mitochondria

Note that chaperones are not involved in folding of proteins in the inter membrane space and that they exist in a low pH environment


Outer membrane

Inner membrane

Mature protein

Inter membrane

Matrix

1.

2.

Alternative Mechanism

There are actually two theories about how the leader operates to localize mtproteins in the inter membrane space:

The first, as shown in the previous slides, involves the whole protein moving into and then out of the matrix

The alternative theory suggests that once the first leader, which targets to the mitochondria is removed, the second leader prevents the protein from ever entering the matrix so it is transported only into the inter membrane space.


Building a Minimally Functional

Nuclear Mitochondrial Gene

Given that a fragment of DNA travels from the mitochondria to the nucleus and is inserted into the nuclear DNA

Nuclear DNA

Control Sequence Signal Sequence Mitochondrial Gene

Additional hurdles may include:

Signal Sequence

Resolution of problems resulting from differences between mitochondrial and nuclear introns

Resolution of problems resulting from differences between mitochondrial and nuclear genetic codes


Additional Requirements

In addition to addition of appropriate control and leader sequences to mitochondrial genes, the following would be needed:

Recognition and transport mechanisms in the cytoplasm

Leader sequence binding receptors

Peptidases that recognize leader sequences and remove them


No Plausible Mechanism Exists

If genes were to move from the mitochondria to the nucleus they would have to somehow pick up the leader sequences necessary to signal for transport before they could be functional

While leader sequences seem to have meaningful portions on them, according to Lewin (1997, p 251) sequence homology between different sequences is not evident, thus there could be no standard sequence that was tacked on as genes were moved from mitochondria to nucleus

Alternatively, if genes for mitochondrial proteins existed in the nucleus prior to loss of genes in the mitochondria, the problem remains, where did the signal sequences come from? And where did the mechanism to move proteins with signal sequences on them come from?


Mitochondrial

Genetic Codes


Variation In Codon Meaning

Lack of variation in codon meanings across almost all phyla is taken as an indicator that initial assignment must have occurred early during evolution and all organisms must have descended from just one individual with the current codon assignments

Exceptions to the universal code are known in a few single-celled eukaryotes, mitochondria and at least one prokaryote

Most exceptions are modifications of the stop codons UAA , UAG and UGA

Organism

Tetrahymena thermophila

A ciliate

Paramecium

A ciliate

Euplotes octacarinatus

A ciliate

Mycoplasma capricolum

A bacteria

Candida

A yeast

Codon/s

UAA UAG

UAA UAG

UGA

UGA

CUG

Common Meaning

Stop

Stop

Stop

Stop serine

Modified Meaning glutamine glutamine cysteine tryptophan leucine

Neutral Non-polar, Polar ©2001 Timothy G. Standish

Variation in Mitochondrial

Codon Assignment

AUA=Met

CUN=Thr

Universal

Code

AAA=Asn AUA=Ile

AAA=Asn

UGA /G=Stop

AUA=Met

NOTE - This would mean

AUA changed from Ile to

Met, then changed back to

Ile in the Echinoderms

AGA/G=Ser AAA must have changed from Lys to

Asn twice

UGA=Trp

UGA must have changed to Trp then back to stop

Differences in mtDNA lower the number of tRNAs needed


Problems Resulting From

Differences in Genetic Codes

Changing the genetic code, even of the most simple genome is very difficult.

Because differences exist in the mitochondrial genomes of groups following changes in the mitochondrial genetic code, mitochondrial genes coding differently must have been transported to the nucleus.

These mitochondrial genes must have been edited to remove any problems caused by differences in the respective genetic codes.


Behe Goes Beyond

Moustraps

In an essay entitled “Intelligent Design theory as a Tool for Analyzing Biochemical Systems,” Michael Behe encourages researchers to go beyond “simple” biochemical systems and to apply Intelligent Design theory to more complex sub-cellular systems. He specifically poses the question:

“Given that some biochemical systems were designed by an intelligent agent, and given the tools by which we came to that conclusion, how do we analyze other biochemical systems that may be more complicated and less discrete than the ones we have so far discussed?”

(Behe, 1998 p 184)


No Modern Examples

Unfortunately for Margulis and S.E.T. [the serial endosymbiotic theory], no modern examples of prokaryotic endocytosis or endosymbioses exist . . . She discusses any number of prokaryotes endosymbiotic in eukaryotes and uses Bdellovibrio as a model for prokaryotic endocytosis.

Bdellovibrios are predatory (or parasitoid) bacteria that feed on E. coli by penetrating the cell wall of the latter and then removing nutrient molecules from E. coli while attached to the outer surface of its plasma membrane. Although it is perfectly obvious that this is not an example of one prokaryote being engulfed by another Margulis continually implies that it is.

P.J. Whitfield, review of “Symbiosis in Cell Evolution,” Biological

Journal of the Linnean Society 18 [1982]:77-78; p 78)


Conclusions

Presence of mitochondrial genes in nuclear DNA reduces the window of time available for mitochondrial acquisition in eukaryotes.

Understanding the structure of mitochondrial genes in the nucleus and how they are expressed makes the transfer of genes from protomitochondria to the nucleus appear complex.

Differences between mitochondrial genetic codes and nuclear genetic codes adds to the complexity of gene transfer between mitochondria and nucleus.

As molecular data accumulates, the endosymbiotic origin of mitochondria appears less probable.


Laboratory


M

PCR of Human mtDNA

Single nucleotide polymorphisms are common in the mtDNA control region. These can be used to identify remains and determine maternal linage due to the maternal inheritance of mitochondria

Single 460 bp mtDNA control region fragment which is polymorphic in sequence, but not size


tRNA Pro

440 bp fragment

Human mtDNA

0

0

1,260

Control region,

D-Loop,

Or hypervariable region

15,971

Left primer

16,411

Right primer

16,569 bp


The Amplified Segment gaaaaagtct t taactccac cattagcacc caaagctaag

Attctaattt aaactattct ctg ttctttc atggggaagc agatttgggt accacccaag tattgactca cccatcaaca accgctatgt atttcgtaca ttactgccag ccaccatgaa tattgtacgg taccataaat acttgaccac ctgtagtaca taaaaaccca atccacatca aaaccccctc cccatgctta caagcaagta cagcaatcaa ccctcaacta tcacacatca actgcaactc caaagccacc cctcacccac taggatacc

Acaaacctac ccacccttaa cagtacatag Tacataaagc catttaccgt acatagcaca ttacagtcaa atcccttctc

Gtccccatgg atgacccccc tcagataggg gtcccttgac caccatcctc c gtga


The Amplified Segment

5’ ct t taactccaccattagcacccaaagctaag…

5’ ttaactccaccattagca

3’

3’ …tcagataggggtcccttgaccaccatcctc c gt

3’ ggaactggtggtaggagg

5’

Following are what I suspect the primers to be:

– Right Primer 5’ ggaggatggtggtcaagg 3’ TM 58.80

– Left Primer 5’ ttaactccaccattagca

3’ TM 49.71


The Amplified Segment

5’ ct t taactccaccattagcacccaaagctaag…

5’ ttaactccaccattagca 3’ cc

3’

…tcagataggggtcccttgaccaccatcctc c gt 3’

3’ ggaactggtggtaggagg 5’

This would up TM and stabilize 3’ end of the primer

Following are what I suspect the primers to be:

– Right Primer 5’ ggaggatggtggtcaagg

3’ TM 58.80

– Left Primer 5’ ttaactccaccattagca 3’ TM 49.71


Human mtDNA Genes

Genes in human (for which numbers are given) and other mammalian mitochondria can be divided into three groups: tRNA genes - 22 rRNA genes - 2

Protein coding genes - 13

Total genes = 37

All protein coding genes are involved in respiration

Aside from the coding portion of genes there is very little additional DNA except in the approximately 1,200 bp control region


Location Strand Length Gene Product

3307..4263

+ 318 ND1 NADH dehydrogenase subunit 1

4470..5513

+ 347

5904..7445

+ 513

ND2 NADH dehydrogenase subunit 2

COX1 cytochrome c oxidase subunit I

7586..8269

+ 227

8366..8572

+ 68

8527..9207

+ 226

9207..9989

+ 260

COX2 cytochrome c oxidase subunit II

ATP8 ATP synthase F0 subunit 8

ATP6 ATP synthase F0 subunit 6

COX3 cytochrome c oxidase subunit III

10059..10406 + 115

10470..10766 + 98

10760..12139 + 459

12337..14148 + 603

14149..14673 - 174

14747..15883 + 378


ND4L NADH dehydrogenase subun 4L




CYTB cytochrome b



Mitochondrial DNA

But as many as received him, to them gave he power to become the sons of God, even to them that believe on his name:

Endosymbiosis and the

Origin of Eukaryotes:

Endosymbiosis

How Mitochondria Resemble Bacteria

How Mitochondria Don’t

Resemble Bacteria

How Mitochondria Don’t

Resemble Bacteria

Archaezoa

Giardia

Giardia

Gene Transport

“All in all then, the host nucleus seems to be a tremendous magnet, both for organellar genes and for endosymbiotic nuclear genes.”

Palmer, 1997

Gene

Expression

The Unlikely Movement of Genes

Between Mitochondria and the Nucleus

Cytoplasm

Cytoplasm

Mitochondrial

Genetic Codes

Laboratory

Related documents

Products

Support

Mitochondrial DNA

But as many as received him, to them gave he power to become the sons of God, even to them that believe on his name:

Endosymbiosis and the

Origin of Eukaryotes:

Endosymbiosis

How Mitochondria Resemble Bacteria

How Mitochondria Don’t

Resemble Bacteria

How Mitochondria Don’t

Resemble Bacteria

Archaezoa

Giardia

Giardia

Gene Transport

“All in all then, the host nucleus seems to be a tremendous magnet, both for organellar genes and for endosymbiotic nuclear genes.”

Palmer, 1997

Gene

Expression

The Unlikely Movement of Genes

Between Mitochondria and the Nucleus

Cytoplasm

Cytoplasm

Mitochondrial

Genetic Codes

Laboratory

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