Engineering the chloroplast genome: techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii

Chloroplast transformation of Chlamydomonas reinhardtii has been accomplished by agitating cell wall-deficient cells in the presence of glass beads and DNA. By using the atpB gene as the selected marker and cells grown in 0.5 mM 5-fluorodeoxyuridine, we have recovered up to 50 transformants per microgram of DNA. This method is easy and does not require specialized equipment, although it is not as efficient as the tungsten particle bombardment method [Boynton, J. E., Gillham, N. W., Harris, E. H., Hosler, J. P., Johnson, A. M., Jones, A. R., Randolph-Anderson, B. L., Robertson, D., Klein, T. M., Shark, K. B. & Sanford, J. C. (1988) Science 240, 1534-1537]. By using particle bombardment, we have developed a cotransformation approach in which spectinomycin-resistant 16S rRNA-encoding DNA is the selected marker, and we have demonstrated that cotransformation of an unselected marker on an independent replicon is very efficient. We have used this strategy (i) to recover transformants with partially deleted atpB genes that could not otherwise have been selected since they did not restore photosynthetic capability to a recipient carrying a more extensive atpB deletion and (ii) to generate specific deletion mutations in a wild-type recipient. This methodology should allow the introduction of any desired change into the chloroplast genome, even in the absence of phenotypic selection, and thus a detailed functional analysis of any chloroplast DNA sequence should be possible.     

Transmission of mitochondrial and chloroplast genomes in crosses of Chlamydomonas.

Physical differences between organelle genomes of the interfertile species Chlamydomonas reinhardtii and Chlamydomonas smithii have been used to demonstrate that sexual zygotes transmit chloroplast and mitochondrial DNA from opposite mating types. Processes responsible can be separated functionally and genetically, although both are controlled by mating type. In vegetative diploids, chloroplast and mitochondrial genomes are transmitted biparentally, but a 1-kilobase insert present in the C. smithii mitochondrial genome spreads unidirectionally to all C. reinhardtii genomes in a manner reminiscent of the intron found in the mitochondrial 21S rRNA gene of omega + strains of yeast.     

Genes for respiratory chain proteins and ribosomal RNAs are present on a 16-kilobase-pair DNA species from Chlamydomonas reinhardtii mitochondria.

We have used heterologous hybridization and DNA sequence analysis to determine whether the 16-kilobase-pair (kbp) DNA from Chlamydomonas reinhardtii mitochondria is the functional equivalent of mtDNA in other eukaryotes. Restriction fragments corresponding to a continuous internal stretch spanning 75% of the 16-kbp DNA have been cloned and mapped, and regions hybridizing with probes specific for the cytochrome oxidase subunit I [CytOx I (acronym COI)] and apocytochrome b (Cyt b) genes of yeast and the mitochondrial 26S and 18S rRNA genes of wheat have been identified. Sequence analysis has verified the presence of CytOx I and the large and small subunit rRNA genes in the C. reinhardtii 16-kbp DNA. In the region of the 16-kbp DNA corresponding to exon 4 in the yeast CytOx I gene, the derived amino acid sequence is 61% and 63% identical with the CytOx I amino acid sequences of yeast and human mitochondria, respectively. Notably, tryptophan is specified by TGG rather than by TGA in this section of the C. reinhardtii CytOx I gene. A probe from the CytOx I region of the 16-kbp DNA hybridizes only with this 16-kbp DNA in Southern blots of total cellular DNA from C. reinhardtii but with a larger DNA species in the total cellular DNA of C. moewusii and C. eugametos--two species that lack a 16-kbp DNA. These observations provide evidence that C. reinhardtii 16-kbp DNA comprises at least part of the mitochondrial genome of this organism and that a homologous DNA exists in other species of Chlamydomonas.     

Nonreciprocal recombination between alleles of the chloroplast 23S rRNA gene in interspecific Chlamydomonas crosses

The inheritance of six polymorphic loci mapping in the rRNA-encoding (rDNA) region of the inverted repeat sequence of chloroplast DNA (cpDNA) was scored in hybrid subclones derived from reciprocal interspecific crosses between the green algae Chlamydomonas eugametos and Chlamydomonas moewusii. In order to enhance the detection of cells that had undergone recombination between parental cpDNAs, hybrids were selected that inherited a chloroplast antibiotic-resistance marker contributed by the mating-type-minus(mt^-) parent, the parent that normally contributes fewer cpDNA molecules. The major findings of this study can be summarized as follows. (i) The majority of the hybrids (14/17) were recombinant for cpDNA markers in the 10-kilobase-pair rDNA region under study. (ii) Only one allele of each polymorphic cpDNA locus was ever detected in the hybrids, thus suggesting that newly recombined rDNA sequences in one copy of the inverted repeat are rapidly spread to the other by a copy-correction mechanism. (iii) Chloroplast streptomycin-resistance (sr-2) and erythromycin-resistance (er-nM1) loci, although showing little or no genetic linkage, were mapped to the 16S and 23S rRNA gene regions of the cpDNA, respectively, by virtue of their perfect coinheritance with polymorphic markers within these genes. (iv) cpDNA markers associated with a putative intron of the C. eugametos 23S rRNA gene were inherited by all 17 hybrids. Such a result is similar to that observed for certain alleles of the large rRNA gene of yeast mitochondria in crosses between ω⁺ and ω^- strains.