Foundations of Biology

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Foundations of Biology

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Title: 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