Download PCB 6528 Exam – Organelle genomes and gene expression

PCB6528 Spring 2011 Chase exam page 1
Name:
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This exam is based on 100 points. It will be worth 64% of your grade for the combined SettlesChase cell biology section.
You may consult books, electronic resources and notes, but NOT other persons as you develop
your answers to this exam.
You may NOT copy text verbatim from your sources; your answers must be phrased in your
own words.
Please return your exam as an attachement to [email protected] by 4 PM on Monday March 1.
Note: As always, there will be alternative answers that can be accepted for full or partial credit
1 [12 pt] Plant mitochondrial and plastid genomes evolved independently from different
bacterial genomes. Consequently, modern day plastid and plant mitochondrial genomes share
some similar features and exhibit some interesting differences. Indicate whether each of the
features below pertain to plastid genomes, plant mitochondrial genomes, both genomes or
neither genome.
Genome Feature
a = plastid genomes
b = plant mitochondrial
genomes
c = both genomes
d = neither genome
Circular maps based upon restriction endonucleases and
overlapping clones
c
Linear and circular molecules of variable size visualized by EM or
FISH
c
Subgenomic forms mediated by recombination through direct
repeats
b
Inversion polymorphisms mediated by recombination through
inverted repeats
c
Genes organized in operons
a
Genetic transformation by homologous recombination
a
Gene order conserved among plant species
a
Gene order highly variable among plant species
b
Gene coding content varies among plant species
c
Genes contain mutations that must be corrected by RNA editing
c
Genes are transcribed by single-subunit, phage-type RNA
polymerases
c
Genes are transcribed by multi-subunit bacterial-type RNA
polymerases
a
PCB6528 Spring 2011 Chase exam page 2
Name:
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2 [38 pt] In 1 page maximum:
a) Define what is meant by retrograde regulation with respect to plant organelles.
[3 pt] Retrograde regulation = changes in nuclear gene expression brought about by signals
from the organelles
b) Based upon class discussion, describe an example of retrograde regulation in plants,
including what is known about upstream events and downstream consequences.
The most discussed example (which I develop below) was the one provided by our discussion
paper. An alternative that was discussed in class is the regulation of nuclear-encoded plastid
proteins via plastid-generated REDOX signals. Examples that we did not discuss in class include
the down-regulation of nuclear-encoded plastid proteins by chlorophyll intermediates and the
down-regulation of B-class MADS box transcription factors in mitochondrial CMS systems.
[5 pt] One example is provided by Shedge et al., Plant Physiol 152:1960. Here, down-regulated
mismatch repair homolog 1 (MSH1) and RECA3 nuclear genes of arabidopsis led to a
mitochondrial retrograde pathway.
[5 pt] Upstream events included: rearranged mitochondrial genomes; increased levels of
mitochondrial transcripts; some novel mitochondrial gene transcripts.
[5 pt] The downstream consequence was up-regulation of a suite of nuclear genes involved in:
response to environmental signals; transcription factors; mitochondrial function; plastid
function; unfolded protein response. The downstream phenotypes included: reduced growth
rate; reduced fertility; striking heat resistance; plants were "primed" to withstand heat stress
c) Choose any strongly affected target of this retrograde signaling that captures your interest. [5
pt] Investigate and briefly describe the nature of the target gene product [5 pt] and the nature
of signals and/or environmental cues that are known to regulate the target gene [5 pt]. Based
upon this knowledge, suggest a hypothesis about the nature of the retrograde signal, and how
this retrograde regulation pathway might be adaptive for plant survival and/or reproductive
success. [5 pt]
Any of the genes listed in Table I or Table II of Shedge et al. are good candidates, as all are upregulated the double mutant prior to heat treatment. For example AT2G21640 encodes protein
located in the mitochondria. Based upon microarray data, it is expressed throughout
Arabidopsis development (e.g. seed, embryo, cotyledon, leaf, and flower)( Schmid et al. Nature
Genetics 37:501;
http://www.arabidopsis.org/servlets/TairObject?name=AT2G21640&type=locus). The
transcript is up-regulated in response to a wide suite of hydrogen peroxide, superoxide and
singlet oxygen generating agents (Gadjev et al. , Plant Physiol. 141: 436). The protein product,
however, was up-regulated in Arabidopsis cell suspension cultures exposed to the superoxide
generating compound menadione, but not to hydrogen peroxide (the common product of
superoxide dismutation) and not to the respiratory complex III inhibitor antimycin A (Sweetlove
et al. Plant J 32:891). Shedge et al. looked at RNA not protein, so up-regulation of ATG21640 in
the msh1 recA3 double mutant indicates a retrograde signal based upon mitochondriaproduced ROS, likely from dysfunction of the respiratory electron transfer chain, but the
specific ROS or ROS-producing site cannot be inferred. ATG21640 is of unknown function, with
no significant BLAST hits to Saccharomyces, Mus, Drosophila, Escherichia, or Chlorophytes, so it
seems to be plant-specific. It is described as "FtsX-like" in Sweetlove et al. FTSX are bacterial
membrane transporter and cell division proteins of the ABC superfamily. Alignment of
PCB6528 Spring 2011 Chase exam page 3
Name:
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AT2G21640 translation product with E. coli FTSX was suggestive but not significant, so function
and further hypotheses about the function and specific adaptive significance require analysis of
mutants. There were a number in the Salk collection.
3 [50 pt] In the table below, list the molecular processes that contribute to organelle gene
expression resulting in fully assembled respiratory and photosynthetic complexes. Indicate
which processes are currently known to involve proteins of the pentatricopeptide repeat (PPR)
class. For those processes which are known to involve PPR proteins, briefly describe an example
by giving 1) the name of the affected organelle gene or protein, 2) the name of the ppr gene,
mutant or protein, and 3) what we know or hypothesize about how the PPR protein functions
to promote the gene expression step in your example. Note, examples where a PPR protein
functions in an indirect manner to facilitate or promote a particular molecular process will be
accepted.
There were many possible examples for these questions, many, but not all, are indicated below.
PPRs have been implicated in most processes and likely it will be all processes as we learn more
and more. Note many PPRs have more than one direct effect and often many indirect effects .
Molecular Process
[6 or more pt]
Known PPR
involvement
(yes or no)
[6 or more
pt]
Example of PPR involvement
[38 pt max]
DNA copy number
no
Transcription
yes
while we did not talk about this in class, several of you found a
couple of interesting examples in the reviews.
1 pep transcribed genes
2 At DG1
3 required for transcription by pep; interacts specifically with
sigma factor 6
(Chi et al. Plant Physiol 147:573)
or
1 pep transcribed genes
2 At PTAC2
3 part of plastid transcription complex; function currently
unknown
(Pfalz et al. Plant Cell 18:176)
RNA
processing/cleavage
yes
1 petB and petD
2-CRP1 (chloroplast RNA processing 1)
3-essential for accumulation of monocistronic transcripts that
result from processing between petB-petD on psbB chloroplast
operon transcript
(Barkan et al. EMBOJ13:3170)
PCB6528 Spring 2011 Chase exam page 4
Name:
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Molecular Process
[6 or more pt]
Known PPR
involvement
(yes or no)
[6 or more
pt]
Example of PPR involvement
[38 pt max]
RNA stability
yes
1 atpH/atpF and psaJ/rpl33
2 PPR10
3 binds RNA 5' to atpH on rps2 operon transcript, and 3' to psaJ
/5' to rpl33 on the pet1 operon transcript; helps to define
processing sites by protecting sites from endonuclease cleavage
and stabilizes processed transcripts by protecting from
degradation by 5' or 3' exonucleases
(Pfalz et al. EMBO J 28:2042)
Intron splicing
yes
While we did not discuss in class a specific example of a PPR
protein required for intron splicing, examples could be easily
found from the required review(Schmitz-Linneweber and Small,
Trends in Plant Sci 13:663)
1- trnG-UCC
2- PPR5
3-binds unspliced trnG-UCC, protects from endonuclease; a
second, direct role in splicing still under investigation
(Williams-Carrier et al. RNA 14:1930)
or
1- Zm rps12
2-PPR4
3- associates with trans-spliced intron 1 of maize plastid rps12
and is required in an as-yet-unknown way for trans-splicing of
this intron
(Schmitz-Linneweber et al. Cell 18:2650)
RNA editing
yes
1-ndhD
2-CRR4 (chloroplast respiratory regulation 4)
3-required for RNA editing that creates the ndhD initiation codon;
how PPRs function in RNA editing is not currently clear; they likely
bind at or near the editing target site; whether they simply make
the target available, actively recruit editing factors or function as
editing factors is not currently known
(Kotera et al. Nature 433:326)
PCB6528 Spring 2011 Chase exam page 5
Name:
key
Molecular Process
[6 or more pt]
Known PPR
involvement
(yes or no)
[6 or more
pt]
Example of PPR involvement
[38 pt max]
Translation
yes
Lots of options here:
1-1 petD
1-2 CRP1 is indirectly essential for translation of petD
1-3 by defining and/or stabilizing the 5' end of the monocistronic
petD transcript; this transcript is required for translation of petD,
likely because the initiation codon is buried in secondary
structure of the di-cistronic petB-petD transcript.
(Schmitz-Linneweber et al. Plant Cell 10:2791)
or
2-1 petA
2-2 CRP1
2-3 we briefly mentioned that CRP1 appears to have an
independent role in translation of petA; CRP1 binds the petA
5'UTR upstream of the initiation site; whether it simply stabilizes
the transcript, makes the AUG available for initiation, or actively
recruits translation factors is not currently known.
or
3-1 ndhD
3-2 CRR4 (chloroplast respiratory regulation 4)
3-3 required indirectly for translation; required for RNA editing
that creates the ndhD initiation codon
Protein complex
assembly and
Protein turnover
yes
1-PETB and PETC
2-CRP1
3-indirectly; CRP1 is required for PETD translation; in the absence
of PETD translation, the PET complex cannot be fully assembled
and translated PETB and PETC subunits are degraded