Evolution of the eukaryotic genomes

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Evolution of the eukaryotic genomes

• Reduced coding content of organelle genomes
• functional gene transfer to nucleus with protein
  targeted back to organelle
• organelles replace prokaryotic features with
  eukaryotic, hybrid or novel features
• Non-functional transfer of DNA
• mit to nucleus plastid to nucleus plastid and
  nucleus to plant mit

Figure 1   Organellar DNA mobility and the
genetic control of biogenesis of mitochondria and
chloroplasts.  From Timmis et al. (2004) Nature
Rev Genet 5123
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Reduced coding content of organelle genomes

Similar trend in the plastids
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RNA Polymerases and promoters
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Differential plastid gene expression based upon
recognition of distinct promoters by NEP and
PEP
(from Hajdukiewicz et al. EMBO J 164041-4048)
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Differential plastid gene expression based upon
sigma subunits
from Lopez-Juez and Pyke Intl J Dev Biol 49557
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Plant organelle RNA metabolism
Like prokaryotes, plant organelle genes are often
co-transcribed as operons In contrast to
prokaryotic transcripts, plant organelle
transcripts Are frequently processed to di or
mono-cistronic transcripts before
translation Frequently contain introns that must
be spliced prior to translation Must undergo an
RNA editing process to restore proper amino acid
coding
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Plant organelle introns
Group I and Group II, defined by characteristic
secondary structures and splicing mechanisms
From Gillham 1994 Organelle Genes and Genomes
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Plant organelle introns
Group I and Group II have distinct splicing
mechansims Group II is the ancestor of the
nuclear intron and the characteristic groupII
intron domains the ancestors of the nuclear
splicosomal RNAs
From Gillham 1994 Organelle Genes and Genomes
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Plant organelle introns
Land plant organelle introns primarily Group
II Characteristic spoke-and-wheel structure
necessary for splicing Some fungal versions are
self-splicing in vitro Trans-acting RNA and/or
protein factors required for splicing in
vivo e.g. maize nuclear mutants encoding
proteins required for splicing Genome
rearrangements have split introns, which then
require trans-splicing The spoke-and-wheel
structure is assembled from separate transcripts
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Trans-splicing Chlamydomonas psaA transcripts
From Gillham 1994 Organelle Genes and Genomes
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Plant organelle RNA editing

Post transcriptional C gt U and less frequently U
gt C genomic coding strand 5 ACG.....
unedited RNA 5 ACG.....
edited RNA 5 AUG....
edited cDNA 5
ATG..... Occurs by enzymatic trans-amination Occ
urs in plastids and plant mitochondria (more
frequently in mitochondria) Occurs primarily in
coding sequences and improves overall
conservation of predicted protein products
Creates initiation codons ACG gt AUG
Creates termination codons CGA gt UGA
Removes termination codons UGA gt CGA
Changes amino acid coding CCA gt CUA (P gt L)
Silent edits
ATC gt ATU Edit sites within the same gene vary
among species. An edit site in one species may
be pre-edited (ie correctly encoded) in the
genomic sequence of another species eg. plastid
psbL gene maize ATGACA.....
tobacco ACGACA.....
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Plant organelle RNA editing
Every land plant lineage except Marchantiid
liverworts from Knoop (2004) Curr Genet
46123

Fig. 2 Several clades in the land plant phylogeny
identified and/or confirmed by molecular data as
monophyletic groups are A angiosperms, S seed
plants, M moniliformopses, E euphyllophytes, L
lycophytes, T tracheophytes. The monophyly of
gymnosperms (G) as a whole and that of a clade
comprising two of its classes, Gnetopsida and
Coniferopsida, is somewhat less well supported.
Likewise, more phylogenetic resolution is needed
for a clade comprising the eusporangiate ferns of
the order Marattiales, the horsetails
(Equisetales) and the leptosporangiate ferns vs a
clade comprising the Ophioglossales and whisk
ferns (Psilotales). The tree shown is
topologically consistent, albeit not always
statistically supported. Several phylogenetic
analyses of organelle genes (unpublished
observations) place hornworts (H) as sister group
to the tracheophytes (node Z), mosses (Ms) as a
sister group to the joint clade (Y) and confirm
marchantiid (ML) and jungermanniid (JL)
liverworts jointly as a sister group to all other
embryophytes (node X)
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RNA editing improves conservation of the
predicted protein (from Mulligan and Maliga 1998)
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RNA editing occurs by enzymatic deamination
from Rajasekhar and Mulligan Plant Cell 51843
from Russell, 1995, Genetics
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RNA editing
Evidence for the importance of cis-guiding
sequences in plant mitochondrial RNA
editing Editing of recombinant or rearranged
mitochondrial genes Recombination breakpoint
immediately 3 to an editing site in rice atp6
did not abolish editing Recombination breakpoint
seven nucleotides 5 to an editing site in maize
rps12 did abolish editing Recombination
breakpoint 21 nucleotides 5 to an editing site
in maize rps12 did not abolish editing
Electroporation of genes into isolated
mitochondria, followed by isolation of
mitochondrial cDNA Editing of mutated coxII gene
demonstrated sequences from 16 to 6 required
for editing
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RNA editing genetic analysis identifies a
trans-acting factor
Figure 3 Analysis of RNA editing in the ndhD
initiation codon. a, Direct sequencing of RTPCR
products containing the ndhD initiation codon.
The psaC and ndhD region is shown schematically.
RNA editing sites are indicated. The restriction
enzyme NlaIII cleaves cDNA derived from edited
molecules. The editing site is so distal in the
transcripts that cDNA was sequenced on the
complementary strand. b, Semi-quantitative
analysis of the extent of RNA editing. RTPCR
products were digested with NlaIII. Fragments
originating from edited and unedited RNA
molecules are indicated. WT, wild type.
from Kotera et al. Nature 433326
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Functional gene transfer from organelle to
nuclear genome

• Gene by gene
• Likely occurs via spliced and edited RNA
  intermediates
• Requires acquisition of a nuclear promoter and
  (often) an organelle targeting pre-sequence
• Evidence for frequent and recent transfers in
  plant lineage
• Results in coding content differences among
  modern-day plant organelle genomes

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Plastid genome transformation
DNA delivery by particle bombardment or PEG
precipitation DNA incorporation by homologous
recombination Initial transformants are
heteroplasmic, having a mixture of transformed
and non-transformed plastids Selection for
resistance to spectinomycin (spec) and
streptomycin (strep) antibiotics that inhibit
plastid protein synthesis Spec or strep
resistance conferred by individual 16S rRNA
mutant Spec and strep resistance conferred by
aadA gene (aminoglycoside adenylyl
transferase) Untransformed callus bleached
transformed callus greens and can be
regenerated Multiple selection cycles may be
required to obtain homoplasmy (all plastid
genomes of the same type)
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Selection for plastid transformants
Figure 1. Generation of tobacco plants with
transgenic chloroplasts A) leaf segments post
bombardment with the aadA gene B) leaf segments
after selection on spectinomycin C) transfer of
transformants to spectinomycin streptomycin to
eliminate spontaneous spectinomycin resistant
mutants D) recovery of homoplasmic spec strep
resistant transformants after multiple rounds of
regeneration on selective medium Bock (2001) J
Mol Biol 312425
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Advantages of plastid genome transformation
High levels of expression Plastid proteins the
most abundant in the world No apparent gene
silencing in plastids Codon preference Bacterial
codon preferences used in the plastid mean that
bacterial genes can be expressed efficiently
without re-engineering codon usage antibiotic
resistance, herbicide resistance, insect
resistance, etc. Containment plastid genes are
not expressed in the pollen (although plastid
genomes may be present in some species)
eliminates pollen toxicity Plastid genomes are
not transmitted through the pollen of many plant
species eliminates pollen transmission of
transgenes to neighboring wild or cultivated
plants Important research tool Precise gene
targeting by homologous recombination