Isolation and Characterization of Chloroplast DNA from Chlorella ellipsoidea

Abstract

A circular DNA molecule was isolated from chloroplasts of Chorella eHlipsoidea. The DNA had a buoyant density of 1.695 grams per cubic centimeter (36% GC) and a contour length of 56 micrometers (175 kilobase pairs). The restriction endonuclease analysis gave the same size. Agarose gel electrophoretic patterns of chloroplast DNA digested by several restriction endonucleases were also presented. The digestion by the restriction enzymes, Hpall, Mspl, SmaI, and XmaI revealed no appreciable methylation at CG sites in chloroplast DNA.The green alga represented by the genus Chlorella includes species that differ considerably in biochemical and physiological characteristics (11). The GC content of the DNAs from different species ranges from 43 to 79% (7). Hybridization of DNAs from different species of Chlorella to one another indicate that there are several distinctively different groups of species within this genus (10). In contrast, studies on cp DNAs' from different strains of Chlorella by means of thermal denaturation and renaturation kinetics showed that this genome was quite homogeneous within this genus (2) and similar to those of other algae such as Chlamydomonas and Euglena (1). Recently, the cp DNAs of Chiamydomonas reinhardii (14, 15) and of Euglena gracilis bacillaris (5) have been characterized. Both cp DNAs were physically mapped with restriction endonucleases and some specific genes (e.g. rRNAs, etc.) were located on these maps. The cp DNA of Chlamydomonas was also shown to be methylated during certain stages of the life cycle of this alga (16). The cp DNAs of these two algae are physically organized quite differently. This raises the question as to the organization of other algal cp DNAs such as those from the genus Chlorella.Recently, Yamada and Sakaguchi (25) induced protoplast formation in Chlorella species and discussed the possibility of isolating intact chloroplasts and cp DNA from the protoplasts. The present study includes the isolation of an intact circular cp DNA of C. ellipsoidea C-87, the measurement of its buoyant density (GC content) and contour length (mol wt), and some restriction endonuclease fragment patterns. This is the first report of the intact isolation of cp DNA from Chlorella. reciprocal shaker with a 16 h light (3,000 lux) and 8 h dark cycle for 4 to 5 d.Preparation of Protoplasts and Chloroplasts. Protoplasts were prepared as previously described from freshly harvested cells (25). For the isolation of chloroplasts, the prepared protoplasts were washed and resuspended in the homogenization medium A (13) with a modification (0.3 M mannitol was replaced by 0.3 M sorbitol). The protoplast suspension (109 cells ml-1) was homogenized at 0°C by a glass homogenizer with a motor-driven Teflon pestle. The homogenate was centrifuged at 1,000 rpm for 5 min to remove unbroken cells and autospores. The supernatant (about 5 ml) was then layered onto a discontinuous density gradient containing 1.0, 1.5, 2.0, and 2.5 M sucrose (17). The gradients were centrifuged at 4°C in an RPS 25 ro...

DNA base modification inChlorella

“…Our whole-cell DNA preparations from C. ellipsoidea contained remarkably high amounts of pt DNA (of the order of 20%) prior to CsC1 density gradient centrifugation. Considering the low DNA G + C content of C. ellipsoidea pt DNA (35'7 mol%; Yamada, 1982), the contaminating pt DNA may also be responsible for the low value determined by Jarvis et al (1992). The total amount of mSdC increased with the G + C content of the DNA.…”

Characterization of a chloroplast DNA sequence from Chlorella ellipsoidea that promotes autonomous replication in yeast

“…Chloroplast DNA from C. ellipsoidea was prepared as described previously (23). Plasmid DNAs for large and small scale preparations were prepared according to Maniatis et al (9).…”

“…Spreading the DNA and preparing, staining and shadowing the grids were performed as previously described (23).…”

“…A characteristic feature of the Chlorella cpDNA is a clustering of GC bases that was previously revealed by digesting the cpDNA with SmaI (recognition sequence CCCGGG). The GC content of this cpDNA is 36°70 (23), so that it is statistically expected to be cleaved with a GC-6 base recognizing restriction enzyme at 6 sites. SmaI virtually digested the cpDNA into many fragments (23 - Fig.…”

Peculiar feature of the organization of rRNA genes of theChlorellachloroplast DNA

“…Chloroplast DNA from C. ellipsoidea was prepared as described previously (9). Plasmid DNAs were prepared according to Maniatis et al (10).…”