The exam was written based on 100 points. Dr. Cline will weight its contribution to your final grade based on the fact that the exam covers material presented in 6 classes. Important – please keep your answers short; confine your answers to the space provided; do not write on the back of any pages ! PCB6528 Spring Exam 2 – Chase Name __KEY__________________________ Page 1 of 4 1 (24 pt) The following statements relate to our class discussion of organelle genomes. For each statement, indicate whether the statement applies to the plastid genome (pt), the plant mitochondrial genome (mit), both plastid and plant mitochondrial genomes (both), or neither of these genomes (none). I was strict on these – right or wrong, no part credit Circular maps based upon DNA sequencing both Linear and circular molecules of various sizes visualized by FISH or EM both Subgenomic forms mediated by recombination through direct repeats mit Inversion isomers mediated by recombination through inverted repeats both Gene order is generally conserved among plant species pt Gene order is highly variable among plant species mit Gene coding content varies among plant species both Genome can be genetically transformed through homologous recombination pt

Modified from Timmis et al. Nat Rev Genet 5:123 A construct that consists of chloroplast sequences (C and D) that flank two selectable marker genes is inserted into the chloroplast genome through homologous recombination, thereby transforming the native plastome into a TRANSPLASTOME (a). One of the selectable genes (aadA) is designed for exclusive expression in the chloroplast and incorporation of this marker confers spectinomycin resistance. The other gene, a neomycin phosphotransferase gene neoSTLS2, includes a nuclear promoter and contains a nuclear intron within the reading frame. NeoSTLS2 confers kanamycin resistance only when it is transposed to the nucleus. Continuous selection of growing leaf cells on spectinomycin medium allows transformed plastomes to be selected and eventually the transplastome entirely replaces the native chloroplast genome, such that all copies of the chloroplast genome contain the two selectable marker genes (b). The transplastomic plants were crossed with wild-type female plants. Because of strict maternal inheritance of tobacco plastids, progeny that contained only wildtype chloroplasts were produced. Selection of progeny seedlings on kanamycin medium allows the detection of the rare cases in which the neo gene has changed its location, such that strong expression is promoted from the nuclear environment (c). Therefore, chloroplast-to-nucleus transposition must have occurred at some stage during the life cycle of the male parent of the seedlings that were recovered on kanamycin plates. The observation that 1 in 16,000 male tobacco gametes contained a segment of chloroplast DNA newly integrated into the nuclear genome was unpredictably high. PCB6528 Spring Exam 2 – Chase Name ____________________________ Page 2 of 4 2 (24 pt) In our discussion paper we saw that plastid genome transformation could be used to genetically mark plastid genomes so that we could follow their fate in grafting experiments. In the experiment described below, genetically marked plastids were used to monitor the transfer of plastid DNA to the nucleus. Explain why this experiment estimates only the frequency of non- functional DNA transfer from plastid to nucleus, rather than the frequency of a plastid gene undergoing a transfer such that its gene function is re-established from the nucleus. (8 pt) In this experiment, the neo gene in the plastid was already fused to a nuclear promoter, so it had only to move to the nucleus to be expressed In a functional gene transfer from plastid to nucleus, the transferred gene would need to acquire a nuclear promoter (10 pt) and also an in-frame targeting signal to direct the protein product back to the plastid (6 pt)

PCB6528 Spring Exam 2 – Chase Name ____________________________ Page 3 of 4 3 (21 pt) The following statements relate to our class discussion of organelle gene expression. For each statement, indicate whether the statement applies to the plastids (pt), the plant mitochondria (mit), both plastid and plant mitochondria (both), or neither organelle (none). I was strict on these – right or wrong, no part credit Genes are transcribed by single-subunit, bacteriophage-type RNA polymerases both Genes are transcribed by multi-subunit, bacterial-type RNA polymerases pt Transcription utilizes nuclear-encoded sigma factors pt Primary transcripts are multi-cistronic operons pt Genes contain group II introns that undergo self-splicing in the organelle none – trick question, see the slide regarding the plastid group II introns and the myriad splicing factors required to properly splice them in the organelle Site-specific RNA binding proteins of the PPR class are involved in multiple gene expression processes both Primary transcripts must undergo C-to-U RNA editing to correct DNA coding errors both

PCB6528 Spring Exam 2 – Chase Name ____________________________ Page 4 of 4 4 (31 pt) In the two experiments shown below, Schult et al (Plant Cell 19:1329) investigated the effects of the hcf173 mutation on the thylakoid proteins of Arabidopsis. What does hcf stand for AND why would the authors expect to see differences in the thylakoid proteins of the hcf mutant compared to wild-type plants? (3 pt) High chlorophyll fluorescence (4 pt) This phenotype results from the inability of the thylakoid- located photosythetic complexes to utilize harvested light energy (2 pt if you said based on the figure below, but I was looking for a general explanation Based on the immunoblotting experiment (below, left): Which thylakoid protein complex appears to be most strongly affected in this mutant? Explain your reasoning. (4 pt) PSII (4 pt) None of the PSII subunits seem to accumulate to even 1/8 wild type levels. PSI proteins also reduced, but not as much Based on the in vivo protein synthesis experiment (below, right), which plastid gene/protein appears to be the one most directly affected by the mutant? Explain your reasoning. (4 pt) D1 protein (4 pt) All of the other PSII proteins appear to be translated at wild-type levels, but D1 translation is much reduced. Based upon your answers above and our class discussions, what aspect of plastid gene expression would you next investigate to determine the primary function of the hcf173 gene with respect to plastid gene expression. Explain your reasoning. (8 pt) anything that is reasonable. I was thinking any aspect of D1 RNA metabolism (transcript accumulation, processing, splicing or editing) to see if there is an RNA-based explanation for the translation phenotype. Some of you focused on protein-protein interactions, or interactions of hcf173 with other RNAs to see why the other PSII subunits were not accumulating, which was also fine. Above - In Vivo Protein Synthesis in Wild- Type Plants and hcf173 Mutants. Newly synthesized proteins of 15-d-old plants were radiolabeled with 35S-Met for 20 min. (A) Wild-type and hcf173 membrane proteins with equivalent amounts of radioactivity (100,000 cpm) or the indicated dilutions were separated by SDS-PAGE and analyzed by fluorography. Left - Immunoblot Analysis of Thylakoid Membrane Proteins from the Wild Type and the Mutant hcf173. Twenty micrograms (WT 1/1 and hcf173), 10 mg (WT 1/2), 5 mg (WT 1/4), and 2.5 mg (WT 1/8) of protein were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Specific proteins were immunodecorated with the indicated antisera.