Foundations of Biology

1
John 112 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

2
Endosymbiosis and the Origin of EukaryotesAre
mitochondria really just bacterial symbionts?

• Timothy G. Standish, Ph. D.

3
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

4
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.

5
Mitochondria
Matrix
Inter membrane space
6
Extranuclear DNA

• Mitochondria and chloroplasts have their own DNA
• This extranuclear DNA exhibits non-Mendalian
  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
• Extra-nuclear 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

7
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 geneome, with import into the
  organelle of nuclear DNA-specified proteins and
  in some cases small RNAs. (Gray et al.,1999)

8
Endosymbiosis
9
Origin of Eukaryotes

• Two popular theories presupposing naturlaism seek
  to explain the origin of membrane bound
  organelles
• 1 Endosymbiosis to explain the origin of
  mitochondria and chloroplasts (popularized by
  Lynn Margulis (Margulis, 1981)
• 2 Invagination of the plasma membrane to form the
  endomembrane system

10
Origin of Eukaryotes

• Two popular theories presupposing naturlaism seek
  to explain the origin of membrane bound
  organelles
• 1 Endosymbiosis to explain the origin of
  mitochondria and chloroplasts (popularized by
  Lynn Margulis (Margulis, 1981)
• 2 Invagination of the plasma membrane to form the
  endomembrane system

11
Origin of Eukaryotes

• Two popular theories presupposing naturlaism seek
  to explain the origin of membrane bound
  organelles
• 1 Endosymbiosis to explain the origin of
  mitochondria and chloroplasts (popularized by
  Lynn Margulis (Margulis, 1981)
• 2 Invagination of the plasma membrane to form the
  endomembrane system

12
Origin of Eukaryotes

• Two popular theories presupposing naturlaism seek
  to explain the origin of membrane bound
  organelles
• 1 Endosymbiosis to explain the origin of
  mitochondria and chloroplasts (popularized by
  Lynn Margulis (Margulis, 1981)
• 2 Invagination of the plasma membrane to form the
  endomembrane system

13
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.

14
How Mitochondria DontResemble Bacteria

• Mitochondria are not always the size or
  morphology of bacteria
• In some Trypanosomes (ie Trypanosoma brucei)
  mitochondria undergo spectacular changes in
  morphology that do not resemble bacteria during
  different life cycle stages (Vickermann, 1971)
• Variation in morphology is common in protistans,
  Considerable variation in shape and size of the
  organelle can occur. (Lloyd, 1974 p1)
• Mitochondrial division and distribution of
  mitochondria to daughter cells is tightly
  controlled by even the simplest eukaryotic cells

15
How Mitochondria DontResemble Bacteria

• Circular mtDNA replication via D loops is
  different from replication of bacterial DNA
  (Lewin, 1997 p441).
• 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 p721,
  888).
• The genetic code in many mitochondria is slightly
  different from bacteria (Lewin, 1997).

16
Archaezoa
17
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

18
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

19
Origin of Gardia

• Gardia and other eukaryotes lacking mitochondira
  and plastids (Metamonada, Microsporidia, and
  Parabasalia ) have been grouped by some as
  Archezoa (Cavalier-Smith, 1983 Campbell et
  al., 1999 pp524-6)
• 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 Gardias fellow Archezoans

20
Origin of Archezoa

• 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 Archezoans
  (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.

21
Origin of ArchezoaMitochondrial Aquisition
22
Origin of ArchezoaGene Transfer and Loss
Lost genetic material
23
Origin of ArchezoaOption 1 - Mitochondrial
Eukaryote Production
24
Origin of ArchezoaOption 2 - Mitochondrial DNA
Loss/Hydrogenosome production
25
Origin of ArchezoaOption 2A -
Mitochondria/Hydrogenosome Loss
26
Gene Transport
27

• All in all then, the host nucleus seems to be a
  tremendous magnet, both for organellar genes and
  for endosymbiotic nuclear genes.
• Palmer, 1997

28
Steps in Mitochondrial AcquisitionThe Serial
Endosymbiosis Theory
29
Steps in Mitochondrial AcquisitionThe Hydrogen
Hypothesis
30
Phylogeny
Cell fusion
31
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 mitochondira to the nucleus exists although
  intraspecies transfer of genes is sometimes
  invoked to explain the origin of other individual
  nuclear genes.

32
Gene Expression
33
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 geneomes 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

34
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
• Mitochondria do not always share the same genetic
  code with the cell they are in
• Mechanisms for transportation of proteins coded
  in the nucleus into mitochondria seem to preclude
  easy movement of genes from mitochondria to the
  nucleus

35
Protein Production Mitochondria and Chloroplasts
36
Protein Production Mitochondria and Chloroplasts
37
Protein Production Mitochondria
Matrix
Inter membrane space
38
Protein Production Mitochondria
Inter membrane space
Matrix
39
Protein Production Mitochondria
40
Protein Production Mitochondria
MLSLRQSIRFFKPATRTLCSSRYLL
Inter membrane space
Matrix
41
Protein Production Mitochondria
Inter membrane space
Matrix
42
Protein Production Mitochondria
43
Protein Production Mitochondria
44
Yeast Cytochrome C Oxidase Subunit IV Leader
Neutral Non-polar Polar Basic Acidic
MLSLRQSIRFFKPATRTLCSSRYLL

• 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 of the
  helix with a rise of 0.54 nm per turn

45
Yeast Cytochrome C1 Leader
MFSNLSKRWAQRTLSKTLKGSKSAAGTATSYFE-KLVTAGVAAAGITAST
LLYANSLTAGA--------------
Neutral Non-polar Polar Basic Acidic

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

46
Protein Production Mitochondria
Matrix
Inter membrane space
47
Protein Production Mitochondria
Inter membrane space
Matrix
48
Protein Production Mitochondria
Inter membrane space
Matrix
49
Protein Production Mitochondria
Inter membrane space
Matrix
50
Protein Production Mitochondria
Inter membrane space
Matrix
51
Protein Production Mitochondria
Inter membrane space
Matrix
52
Protein Production Mitochondria
Inter membrane space
Matrix
53
Protein Production Mitochondria
Inter membrane space
Matrix
54
Protein Production Mitochondria
Inter membrane space
Matrix
55
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
Inter membrane space
Matrix
56
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.

57
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

• Additional hurdles may include
• Resolution of problems resulting from differences
  between mitochondrial and nuclear introns
• Resolution of problems resulting from differences
  between mitochondiral and nuclear genetic codes

58
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

59
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, p251)
  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?

60
Mitochondrial Genetic Codes
61
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

62
Variation in Mitochondrial Codon Assignment

• NOTE - This would mean AUA changed from Ile to
  Met, then changed back to Ile in the Echinoderms

• AAA must have changed from Lys to Asn twice

• UGA must have changed to Trp then back to stop

• Differences in mtDNA lower the number of tRNAs
  needed

63
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.

64
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 p184)

65
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 198277-78 p. 78)

66
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.

67
Laboratory
68
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
69
Human mtDNA
70
The Amplified Segment

• gaaaaagtct ttaactccac cattagcacc caaagctaag
• Attctaattt aaactattct ctgttctttc 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 cgtga

71
The Amplified Segment

• 5ctttaactccaccattagcacccaaagctaag
• 5ttaactccaccattagca3
• 3tcagataggggtcccttgaccaccatcctccgt
• 3ggaactggtggtaggagg5
• Following are what I suspect the primers to be
• Right Primer 5ggaggatggtggtcaagg3 TM 58.80
• Left Primer 5ttaactccaccattagca3 TM 49.71

72
The Amplified Segment
5ttaactccaccattagca3
3ggaactggtggtaggagg5

• Following are what I suspect the primers to be
• Right Primer 5ggaggatggtggtcaagg3 TM 58.80
• Left Primer 5ttaactccaccattagca3 TM 49.71

73
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

74

• Location Strand Length Gene Product
• 3307..4263 318 ND1 NADH dehydrogenase
  subunit 1
• 4470..5513 347 ND2 NADH dehydrogenase
  subunit 2
• 5904..7445 513 COX1 cytochrome c oxidase
  subunit I
• 7586..8269 227 COX2 cytochrome c oxidase
  subunit II
• 8366..8572 68 ATP8 ATP synthase F0 subunit
  8
• 8527..9207 226 ATP6 ATP synthase F0 subunit
  6
• 9207..9989 260 COX3 cytochrome c oxidase
  subunit III
• 10059..10406 115 ND3 NADH dehydrogenase
  subunit 3
• 10470..10766 98 ND4L NADH dehydrogenase
  subun 4L
• 10760..12139 459 ND4 NADH dehydrogenase
  subunit 4
• 12337..14148 603 ND5 NADH dehydrogenase
  subunit 5
• 14149..14673 - 174 ND6 NADH dehydrogenase
  subunit 6
• 14747..15883 378 CYTB cytochrome b

75
(No Transcript)