Cyanidioschyzon merolae

Cyanidioschyzon merolae is a small (2μm), club-shaped, unicellular haploid red alga adapted to high sulfur acidic hot spring environments (pH 1.5, 45 °C). The cellular architecture of C. merolae is extremely simple, containing only a single chloroplast and a single mitochondrion and lacking a vacuole and cell wall. In addition, the cellular and organelle divisions can be synchronized. For these reasons, C. merolae is considered an excellent model system for study of cellular and organelle division processes, as well as biochemistry and structural biology. The organism's genome was the first full algal genome to be sequenced in 2004; its plastid was sequenced in 2000 and 2003, and its mitochondrion in 1998. The organism has been considered the simplest of eukaryotic cells for its minimalist cellular organization. Originally isolated by De Luca in 1978 from the solfatane fumaroles of Campi Flegrei (Naples, Italy), C. merolae can be grown in culture in the laboratory in Modified Allen’s medium (MA) or a modified form with twice the concentration of some elements called MA2. Using MA medium, growth rates are not particularly fast, with a doubling time (the time it takes a culture of microbes to double in cells per unit volume) of approximately 32 hours. By using the more optimal medium MA2, this can be reduced to 24 hours. Culturing is done at 42 °C under white fluorescent light with an approximate intensity of 50 µmol photons m−2 s−1 (µE). However, under a higher light intensity of 90 µE with 5% CO2 applied through bubbling, the growth rate of C. merolae can be further increased, with a doubling time of approximately 9.2 hours. Higher light is not necessarily beneficial, as above 90 µE the growth rate begins to decrease. This may be due to photodamage occurring to the photosynthetic apparatus. C. merolae can also be grown on gellan gum plates for purposes of colony selection or strain maintenance in the laboratory. C. merolae is an obligate oxygenic phototroph, meaning it is not capable of taking up fixed carbon from its environment and must rely on oxygenic photosynthesis to fix carbon from CO2. The 16.5 megabase pair genome of C. merolae was sequenced in 2004. The reduced, extremely simple, compact genome is made up of 20 chromosomes and was found to contain 5,331 genes, of which 86.3% were found to be expressed and only 26 contain introns, which contained strict consensus sequences. Strikingly, the genome of C. merolae contains only 30 tRNA genes and an extremely minimal number of ribosomal RNA gene copies, as shown in the genome comparison table. The reduced nature of the genome has led to several other unusual features. While most eukaryotes contain 10 or so copies of the dynamins required for pinching membranes to separate dividing compartments, C. merolae only contains two, a fact that researchers have taken advantage of when studying organelle division. Although possessing a small genome, the chloroplast genome of C. merolae contains many genes not present in the chloroplast genomes of other algae and plants. Most of its genes are intronless. As is the case with most model organisms, genetic tools have been developed in C. merolae. These include methods for the isolation of DNA and RNA from C. merolae, the introduction of DNA into C. merolae for transient or stable transformation, and methods for selection including a uracil auxotroph that can be used as a selection marker. Several methods, derived from cyanobacterial protocols, are used for the isolation of DNA from C. merolae. The first is a hot phenol extraction, which is a quick extraction that can be used to isolate DNA suitable for amplification by DNA polymerase chain reaction (PCR), wherein phenol is added to whole cells and incubated at 65 °C to extract DNA. If purer DNA is required, the CTAB (Cetyl trimethyl ammonium bromide) method may be employed. In this method, a high-salt extraction buffer is first applied and cells are disrupted, after which a chloroform-phenol mixture is used to extract the DNA at room temperature. Total RNA may be extracted from C. merolae cells using a variant of the hot phenol method described above for DNA. As is the case for DNA and RNA, the protocol for protein extraction is also an adaptation of the protocol used in cyanobacteria. Cells are disrupted using glass beads and vortexing in a 10% glycerol buffer containing the reducing agent DTT to break disulfide bonds within proteins. This extraction will result in denatured proteins, which can be used in SDS-PAGE gels for Western blotting and Coomassie staining.