The goal of this research was to characterize the organellar genomes of Chlorella and other microalgae. As organellar DNA is thought to be highly conserved evolutionarily, the idea was to use similarities or differences between chloroplast or mitochondrial DNA as a measure of the taxomonic relatedness of algal strains. This information could be useful for experiments involving somatic cell fusion or gene transfer, as these procedures would likely have a higher chance of success between more closely related strains. Studies of the organellar genomes could also lead to the identification of promoters or replication origins that could be used to develop vectors for algal transformation. Due to the lack of significant progress on the first three goals, Dr. Meints concentrated the efforts of his laboratory on this project for the last 2 years of the subcontract.

The first step was to develop methods for isolation of chloroplast and mitochondrial DNA from Chlorella N1a. Based on protocols used for higher plants, Dr. Meints exploited the differences in the C/G content between chloroplast DNA and nuclear DNA to separate the two genomes using density gradient centrifugation. Chloroplast DNA was identified by hybridization with heterologous chloroplast DNA markers. The chloroplast genome of Chlorella N1a was found to be circular, containing approximately 120 kbp of DNA. A restriction map of the chloroplast genome was produced and several genes were localized on the map by hybridization with chloroplast gene sequences from maize. Most chloroplast genomes contain two inverted repeats, each of which contains a copy of the 23S, 16S, and 5S ribosomal RNA genes. These repeats are flanked by a short and long single copy DNA region. Although Dr. Meints initially reported that Chlorella N1a chloroplast DNA contained this inverted repeat structure (Meints 1987), a subsequent article reported that the chloroplast genome of Chlorella N1a contains only a single copy of the ribosomal RNA gene region (Schuster et al. 1990b). This result was confirmed by Dr. Meints via a recent personal communication. Although most other green algae, including other chlorellans, contain chloroplast DNA similar to that commonly seen in most higher plants, i. e., containing two inverted repeats, this unusual chloroplast structure has been seen in two legumes (peas, broad beans), conifers, some red algae, and in at least one other green alga, Codium.

Restriction analysis of chloroplast DNA from several exsymbiont and free-living strains of Chlorella showed variations between the strains that indicate genetic divergence and that suggest gene transfer and cell fusion between these species may be problematic. The results suggest that chloroplast DNA structure may be a useful taxonomic parameter, but more study is needed before definite conclusions on algal taxonomy or cell-cell compatability based on chloroplast DNA structure can be made.

Isolating mitochondrial DNA from Chlorella N1a was technically problematic, and the mitochondrial genome isolated was first presumed to be a plasmid. Unlike some species in which mitochondrial DNA has a G/C content similar to that of nuclear DNA, in Chlorella N1a, the mitochondrial DNA had a low G/C content similar to that of chloroplast DNA, and the two genomes banded very closely on the density gradients. As with the chloroplast DNA,

heterologous probes were used to identify the mitochondrial DNA and to localize specific mitochondrial genes on the restriction map. The gene organization in the Chlorella N1a mitochondrial DNA was similar to that in higher plants, and distinct from the organization of mitochondrial genes in animals and fungi. It has been proposed that mitochondria in plants and green algae originated from a separate endosymbiotic event as compared to animals and fungi. This is supported by Dr. Meints’ results.

Although not included in the original Statement of Work, Dr. Meints also reported under this task other related research efforts in his laboratory toward the development of a genetic transformation system for microalgae. Libraries were prepared from Chlorella N1a nuclear DNA and DNA from the algal virus. The goal of this project was to identify DNA sequences that could be used to develop transforming vectors, such as origins of replication, regulatory regions for gene expression, or algal genes to use in selectable marker systems. A library of the viral DNA was prepared in a lambda vector, which allowed for the sequencing of the viral genome and studies of viral gene stucture and expression. This work led to several significant discoveries that were published after SERI funding stopped, including the cloning of the major viral capsid protein (Graves and Meints, 1992), and the identification of a viral gene promoter that also functioned in higher plants (Mitra and Higgins, 1994).

Dr. Meints’ laboratory also made several attempts to produce a library of Chlorella nuclear DNA, with little success. This appeared to be due to modification (probably methylation) of the algal DNA that resulted in degradation of the DNA by the bacterial host used for library construction. Several ways around this problem were proposed, including the use of a yeast cloning system or the use of a bacterial host that did not contain the enzymes for degradation of methylated DNA. A cDNA library was produced successfully before the end of the SERI-funded research efforts.

Dr. Meints and his coworkers and collaborators produced a large quantity of data during the 4 years of SERI-funded research and during the following years. They made significant contributions to the study of the biology, biochemistry, and molecular biology of a eukaryotic algal virus, and to the biology and molecular biology of the algal hosts, particularly with respect to the algal organellar genomes. Unfortunately, because of the specificity of the virus/algal interactions, the results obtained were not directly applicable to the development of a transformation system for the oleaginous algal strains of interest to NREL. The research also generated some valuable technical information, regarding toxicity of microalgae to common osmotica, construction of genomic DNA libraries, and organellar genome isolation, which could be useful for further studies of algal molecular biology and the development of genetic engineering techniques. The studies of the algal virus also resulted in the identification of a new restriction endonuclease (Jin et al. 1994) and a new adenine methyltransferase (Stefan et al. 1991), as well as a viral promoter sequence that can function in plants (Mitra and Higgens 1994).