The chloroplast genome was the first plant genome characterized. Its small size and limited number of repeated elements made it a prime candidate for characterization. Its abundance in foliar tissue made it easy to isolate. The inverted repeat is an interesting feature of the chloroplast genome, and is found in most species outside the Leguminosae. Beyond that, what was a tour de force in the mid-1980's has now become trivial. A healthy BAC is about the same size as a chloroplast genome, and sequencing of BACs is now a standard lab procedure. If time permits, we will do partial BAC sequencing before the class is out.
Once isolated, characterization of plastid genome structure required restriction and developing of overlapping restriction site maps. The plastid genome was determined to be circular through construction of complete genome maps. Complete chloroplast genome sequences have been reported for maize, rice, liverwort, Chlamydomonas, Euglena and several other interesting plant species.
Study question: from a series of single and double restriction
enzyme digests, can you construct a map of a circular DNA molecule?
If you're interested in a Cook's tour of plastid function,
try this out.
Study questions: How do you determine maternal inheritance? (I will give you a group of datasets in which dominance, codominance, epistasis, maternal and paternal inheritance patterns are demonstrated. Can you discern one from another? Can you determine when further tests are required, and can you tell me what sorts of further tests would be helpful?) How does maternal inheritance differ from cytoplasmic inheritance? What kinds of markers are appropriate in monitoring cytoplasmic or maternal inheritance? Why is maternal inheritance of chloroplasts so common?
The first plant chloroplast genome completely sequenced was the liverwort (Marchantia polymorpha) genome (Ohyama et L. Science 322:572-574). The liverwort ct genome contains 121,024 bp which contains two 10,058 bp inverted repeats, separated by a small single copy region (19,813 bp) and a large single copy region (81,095bp).The authors used standard sequence analysis tools to 1) deduce open reading frames (study question: what is an open reading frame, and how do you deduce it?), then compared homology of all ORFs with the E. coli genome database (why E. coli?).
The chloroplast genome contains genes encoding the major ribosomal RNAs, and about half of the required ribosomal proteins. All required tRNAs are encoded, and five are contained within the inverted repeat. No major chloroplast function has been ascertained which can be fully accomplished by proteins encoded solely by the chloroplast. All chloroplast functions require imported proteins encoded by the nuclear genome.
The Lee Papers: Given that we were lucky in finding polymorphisms which could help us track both YGS and ALS, how would you approach the problem today? How would you identify the mutation(s) responsible for the mutation(s) responsible for the phenotypes?
Perhaps the only really practically important variable character associated with chloroplasts is atrazine resistance . This is conditioned by a single base change which results in a simple amino acid substitution (serine->glycine) in the PsbA gene. This apparantly reduces the binding efficiency of atrazine, rendering the plant resistant.
Somatic Hybrids: It's relatively simple to prepare somatic hybrid
plant cells. Take two plants which can be regenerated from protoplasts,
fuse the protoplasts and regenerate plants. This has been ensured by employing
selecting markers in each cell line. Atrazine resistance was utilized (as
was light sensitivity) in Robertsn, Palmer, Earle and Mutschler (1987)
Tag 74:303-309.
The other side of luck in research: the plant was female sterile (although male fertile). Study question: Why was this a particularly poor turn of luck?
Atrazine resistance is a ct encoded trait. The plant was atrazine resistant. When differences between the campestris and oleraceae ct. genomes were evaluated, all five of the campestris (atrazine resistant) markers were present. The chloroplast genome was non-recombinant.
Day and Ellis (1984) Chloroplast DNA deletions associated with
wheat plants regenerated from pollen: Possible basis for maternal inheritance
of chloroplasts. Cell(39)359-368.
In order to see a ct band from a small amount of tissue, it was necessary to isolate total nucleic acids, digest with PstI and try to regenerate a circular map by repetitive probing. Circular maps were generated. What did they show? What mechanisms were proposed to explain the observed deletions?
Ogihara et al., 1988. Intramolecular recombination of chloroplast genome mediated by short direct-repeat sequences in wheat species. PNAS 85:8573-8577.
Three wheat relatives, T. Aestivum, AE. Crassa and Ae. Squarossa show
small, simple deletion differences which differentiate their ct genomes.
Maps were constructed in each species. The deletions were characterized,
and the regions surrounding (and in the non-deleted species the deleted
interval) were sequenced. The sequences were aligned. As the figure demonstrates
(Fig. 2), if you assume sequence slippage and deletion of the 'loop', the
variation among genomes is well-explained.
well-conserved, low rate of mutations
excellent cladistic/phylogenetic tool
multi-genomed organelle
multiple chloroplasts per cell
absolute symbiote- no completely self-encoded functions
Several genomes have been completely sequenced.
The Mitochondrial Genome
Jeff Palmer once again came to our assistance and sorted out the mitochondrial genome. It's multicircular, intragenomic recombination occurs in 'real time', resulting in maps which are initially pretty confusing. Brassica species have pretty simple genomes- a major curcular chromosome which undergoes internal recombination to form two circular sub-genomic chromosomes.
Interesting aspects of mitochondrial genome genetics include cytoplasmic
male sterility and repeated hints at adaptationally-useful
variation.