Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase.
We have developed a nuclear transformation system for Chlamydomonas reinhardtii, using micro-projectile bombardment to introduce the gene encoding nitrate reductase into a nit1 mutant strain which lacks nitrate reductase activity. By using either supercoiled or linear plasmid DNA, transformants were recovered consistently at a low efficiency, on the order of 15 transformants per microgram of plasmid DNA. In all cases the transforming DNA was integrated into the nuclear genome, usually in multiple copies. Most of the introduced copies were genetically linked to each other, and they were unlinked to the original nit1 locus. The transforming DNA and nit+ phenotype were stable through mitosis and meiosis, even in the absence of selection. nit1 transcripts of various sizes were expressed at levels equal to or greater than those in wild-type nit+ strains. In most transformants, nitrate reductase enzyme activity was expressed at approximately wild-type levels. In all transformants, nit1 mRNA and nitrate reductase enzyme activity were repressed in cells grown on ammonium medium, showing that expression of the integrated nit1 genes was regulated normally. When a second plasmid with a nonselectable gene was bombarded into the cells along with the nit1 gene, transformants carrying DNA from both plasmids were recovered. In some cases, expression of the unselected gene could be detected. With the advent of nuclear transformation in Chlamydomonas, it becomes the first photosynthetic organism in which both the nuclear and chloroplast compartments can be transformed.
Kindle
K L
KL
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
Schnell
R A
RA
Fernández
E
E
Lefebvre
P A
PA
D002467:Cell Nucleus / Q000378:metabolism*; D002696:Chlamydomonas / Q000201:enzymology / Q000235:genetics*; D004247:DNA / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005796:Genes; D008938:Mitosis; D009565:Nitrate Reductases / Q000235:genetics* / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D015183:Restriction Mapping; D014170:Transformation, Genetic
D004247:DNA; D009565:Nitrate Reductases